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Biochemical characterization of a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1

Ubolsree Leartsakulpanich

Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirement for the degree of

Doctor of philosophy in Biochemistry

Dr. James G. Ferry, Chair Dr. John L. Hess Dr. Eugene M. Gregory Dr. Timothy J. Larson Dr. Dennis R. Dean Dr. Robert H. White

June 1999 Blacksburg, Virginia Keywords: Iron-sulfur flavoprotein, Methanosarcina thermophila Copyright 1999, Ubolsree Leartsakulpanich

Ubolsree Leartsakulpanich

ABSTRACT

The iron-sulfur flavoprotein (Isf) from the acetate utilizing methanoarchaeon Methanosarcina thermophila was heterologously produced in Escherichia coli, purified to homogeneity, and characterized to determine the properties of the iron-sulfur cluster and FMN. Chemical and spectroscopic analyses indicated that Isf contained one 4Fe-4S cluster and one FMN per monomer. The midpoint potentials of the [4Fe-4S]2+/1+ center and FMN/FMNH2 redox couple were -394 and -277 mV respectively.

The deduced amino acid sequence of Isf revealed high identity with Isf homologues from the CO2 reducing methanoarchaea Methanococcus jannaschii and Methanobacterium thermoautotrophicum. Extracts of H2-CO2-grown M. thermoautotrophicum cells were able to reduce Isf from M. thermophila using either H2 or CO as the reductant. Addition of ferredoxin A to the reaction further stimulated the rate of Isf reduction. These results suggest that Isf homologues are coupled to ferredoxin in electron transfer chains in methanoarchaea with diverse metabolic pathways.

Reconstituted systems containing carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS), ferredoxin A, Isf, and the designated electron carriers (NAD, NADP, F420, and 2-hydroxyphenazine) were used in an attempt to determine the electron acceptor for Isf. Isf was unable to reduce any of these compounds. Furthermore, 2-hydroxyphenazine competed with Isf to accept electrons from ferredoxin A indicating that ferredoxin A is a more favorable electron partner for 2-hydroxyphenazine. Thus, the physiological electron acceptor for Isf is unknown.

Amino acid sequence alignment of Isf sequences revealed a conserved atypical cysteine motif with the potential to ligate the 4Fe-4S cluster. Site-directed mutagenesis of the cysteine residues in this motif, and the two additional cysteines in the sequence, was used to investigate these cysteine residue as ligands for coordinating the 4Fe-4S

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center of Isf. Spectroscopic and biochemical analyses were consistent with the conserved cysteine motif functioning as ligating the 4Fe-4S center. Redox properties of the 4Fe-4S and FMN centers revealed a role for the 4Fe-4S center in the transfer of electrons from ferredoxin A to FMN.

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FORWARD

This dissertation focuses on the characterization of the heterologously produced iron-sulfur flavoprotein from Methanosarcina thermophila by different approaches. Chapters 1 and 2 are intended to serve as an introduction to biological methanogenesis and iron-sulfur proteins. Chapters 3 and 4 describe the research pertaining to the characterization of iron-sulfur flavoprotein and a summary is presented in Chapter 5. The studies described in Chapters 3 and 4 have or will be published as follows:

Becker D.F., Leartsakulpanich U., Surerus K.K., Ferry J.G., and Ragsdale S.W. 1998. Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol Chem. 273:26462-26469

Leartsakulpanich U, Antokine M.L., Golbeck J.H., and Ferry J.G. A novel [4Fe4S] iron-sulfur cluster binding motif in the iron-sulfur flavoprotein of Methanosarcina thermophila (manuscript in preparation).

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ACKNOWLEDGEMENT

I would like to express my gratitude to my thesis advisor, Dr. James G. Ferry, for giving me the opportunity, financial support, encouragement and guidance in my academic career. This dissertation would be impossible with out his guidance and insight toward my study. I would like to thank my committee members, Dr. T.J. Larson, Dr. D.R. Dean, Dr. E.M. Gregory, and Dr. J.L. Hess, for their interest in my research and their flexibility and understanding of my situation as an out of state student. In addition, I would like to thank Dr. R.H. White for being substituted for Dr. Dean as another thesis examining committee. I thank Dr. D.F. Becker, Dr. S.W. Ragsdale, and Dr. K.K. Surerus for their contributions to the characterization of the iron-sulfur flavoprotein (Isf). I thank M.L. Antokine and Dr. J.H. Golbeck for their assist on EPR experiments with Isf variants. I gratefully acknowledge the financial support from MOSTE, Thailand throughout my 5 years in the US.

I also would like to thank all the postdocs and students in Dr. Ferry's lab. Cheryl Ingram Smith (who helped me during my research rotation and has been very helpful since, also for her compassion and caring), Birgit Alber (who was another Hoakie, special soul-mate, my volleyball coach, and my driver when I wanted to head south), Kerry Smith and Rob Barber (for their muscles and advice and thousands of suggestion), Madeline Rasche, Kavita Singh-Wissman, and Julie Maupin-Furlow (for their kindness and patience in teaching me techniques), Sean O'Hearn (for let me know that being a biochemist is better than being a chef, Really?) and Mike Painter, Tong Zhao, Rebecca Miles, Christie Brosius, Birthe Borup, Prabha Iyer, Brian Tripp, Laura Lierman, and Mark Signs, for their friendship, English teaching, and support (especially when I cannot smile). My appreciation goes to the secretary of the department at VA Tech, Mary Jo Smart, and several secretaries of the Ferry lab, Vonni Kladde, and Carol DeArmitt, for taking care of things promptly, efficiently, and conveniently for me.

I want to extend my appreciation to my ex-roommates at VATech, Kitsiri Kaewpipat and Chanpen Chanchao, together with Sunitiya Thuannadee, Charaspim

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Boonyanan, and Matt Mamorino who made the years in Blacksburg an incredibly wonderful experience for me. Also, Somboon Kiratiprayoon, Patcharin Poosanaas, Amin Tanuminhadjo, Bill and Barb Saxton, Joanie Zhoa and HenSiong Tan, Ari and Purwadi Purwasumato Venyi Hoa, Joy Wang, Maki Murata, Kerwin Foster, and all other members of ICF, who supply the happiness at PSU.

Finally and the most important, I would like to express my deep gratitude to my family, Khajon (for taking all the burden off my shoulders and letting me continue my studies), Sirinthip (for her compassion, consideration, and love), Paramate ( for his sharing, and prompt assistance with seemingly ceaseless energy), and particularly to my Mom and Dad (for their unconditional and endless love, supporting guidance, generosity, sweetness, and always always being there for me); without their inspiration, encouragement, motivation, and optimism, I would not be able to come this far. Thanks again to all.

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TABLE OF CONTENTS

Page Title page Abstract Forward Acknowlegement Table of contents List of tables List of figures Introduction Chapter 1: Methanogenesis I. Microbiology Methanoarchaea Growth substrates Ecology II. Biochemistry Coenzymes CO2-reduction pathway Acetate fermentation pathway III. Bioenergetics and electron transport References Chapter 2: Iron-sulfur proteins I. Introduction II. Structural and properties of clusters 1[Fe] cluster type [2Fe-2S] cluster type [3Fe-4S] cluster type [4Fe-4S] cluster type III. Ligation of iron-sulfur clusters IV. Function of iron-sulfur centers

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i ii iv v vii ix x xiii 1 1 1 1 2 6 6 8 10 14 18 32 32 32 33 34 34 35 39 41

TABLE OF CONTENTS (cont.) Page A. Electron transfers B. Catalysis C. Regulatory role D. Iron storage role E. Structural role V. Iron-sulfur cluster assembly in proteins References Chapter 3: Objectives Chapter 4: Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila Abstract Introduction Materials and Methods Results Discussion Acknowledgement References Chapter 5: A novel [4Fe-4S] iron-sulfur cluster binding motif in the iron-sulfur flavoprotein of Methanosarcina thermophila Abstract Introduction Experimental procedures Results Discussion Acknowledgement References Chapter 6: Summary and future directions Curriculum Vista 88 88 89 91 93 118 120 121 124 126 57 57 57 59 63 81 84 85 41 42 43 44 44 45 46 55

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LIST OF TABLES

Page Chapter 1 Table 1. Substrates for methanogenesis Chapter 5 Table 1. EPR properties of wild-type Isf and variants Table 2. Rates for reduction of FMN in wild type Isf and variants 115 117 3

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LIST OF FIGURES

Page Chapter 1 Figure 1. Microbial food chain Figure 2. Structure of the coenzyme of the coenzymes involved in methanogenesis Figure 3. Methanogenesis from CO2 reduction pathway Figure 4. Proposed pathway for acetate conversion to CO2 and CH4 in Methanosarcina thermophila Chapter 2 Figure 1. Structures and properties of the structurally characterized iron-sulfur centers that are involved in biological system Figure 2. Arrangement of residues involved in coordination of [2Fe-2S] (A), [4Fe-4S] or 2[4Fe-4S] (B), [3Fe-4S] or [4Fe-4S] (C), and 2[4Fe-4S] or [3Fe-4S] plus [4Fe-4S] (D) clusters Chapter 4 Figure 1. Corrected nucleic acid sequence and predicted amino acid sequence of isf from M. thermophila Figure 2. EPR spectroscopy of the [4Fe-4S] cluster in Isf poised at various redox potentials in 50 mM potassium phosphate buffer (pH 7.0) Figure 3. Semilogarithmic plot of P1/2 versus 1/T, which shows a linear relationship according to equation P1/2 = Aexp (-/kT) Figure 4. Mössbauer spectra recorded at 100 K. A) oxidized Isf protein. B) reduced Isf protein Figure 5. Mössbauer spectra of reduced Isf protein recorded at 4.2 K and 450 G applied parallel (A) or 450 G applied perpendicular (B) to the beam

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7 9

13

36

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64

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67

69

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Figure 6. Potentiometric titration of the FMN in Isf (3.2 µM) in 50 mM potassium phosphate buffer (pH 7.0) at 20o C (curves 1-7, fully oxidized, -262, -272, -281, -290, -305, and ­342 mV respectively). Inset, Nernst plot of the potentiometric data Figure 7. EPR spectrum of Isf (170 µM dimer) was recorded at 10 K following incubation for 17 min with CO and CODH (0.5 µM) at 25o C) Figure 8. A fit of the Isf midpoint potential data to a theoretical curve generated from the Nernst equation for two redox centers with reduction potentials of ­277 mV (n = 2) and ­394 mV (n = 1) Figure 9. Time course for reduction of methanophenazine with Isf from M. thermophila Figure 10. Multiple amino acid sequence alignment of Isf from M. thermophila with sequences deduced from open reading frames identified in the genomic sequences of M. jannashii and M. thermoautotrophicum Figure 11. Time course for reduction of Isf with extract from M. thermoautotrophicum Figure 12. Time course for reduction of Isf with extract from M. thermoautotrophicum Figure 13. Proposed electron transport pathway for oxidation of CO or the carbonyl group of acetyl-CoA Chapter 5 Figure 1. Multiple amino acid sequence alignment of Isf from M.thermophila (MST) with sequences deduced from open reading frames identified in the genomic sequences of M. jannaschii (MCJ), M. thermoautotrophicum (MBT), Archaeoglobus fulgidus (AF), Chlorobium vibrioforme (CV), Chlorobium tepidum (CT), and Clostridium difficile (CD).

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76

78

79

80

82

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Figure 2. Coomassie blue stained native PAGE of wild type Isf and variants Figure 3. UV-visible absorption spectra of as-purified, denatured, and reconstituted wild type Isf Figure 4. UV-visible absorption spectra of wild type Isf and alanine variants Figure 5. UV-visible absorption spectra of wild type Isf and serine variants Figure 6. EPR spectra of reduced wild type Isf with different processes to recover iron-sulfur center Figure 7. EPR spectra of C16X (X = A or S) Figure 8. EPR spectra of reduced C180A with different reconstitution processes Figure 9. EPR spectrum of reduced C180S Figure 10. EPR spectrum of as-purified C50A Figure 11. EPR spectra of as-purified C59A Figure 12. EPR spectrum of reduced C47A Figure 13. EPR spectrum of reduced C53A Figure 14. EPR spectrum of reduced C47S Figure 15. EPR spectrum of reduced C50S Figure 16. EPR spectrum of reduced C53S 105 106 107 108 110 111 112 113 114 102 104 101 100 99 97

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INTRODUCTION

Methanogenesis is a prominent process in the biological world, in which it represents the final step in the carbon cycle in anaerobic environment. Methanoarchaea have a major impact on the environment and human activities. More than 109 tons of methane have been released into the atmosphere. Methane is produced by two major pathways. The first is the CO2 reduction pathway in which CO2 is reduced to methane using electrons derived from either H2 or formate. Acetate is a key product in the decomposition of organic compounds and is the primary substrate for methane production with two-thirds of all biological methane derived the methyl group of acetate. However, only species of the genera Methanosarcina and Methanothrix are known to convert acetate to methane and CO2. The study of methanogenesis has made an enormous impact in many areas of physiology, ecology, biochemistry, molecular biology, and evolution. Methanosarcina thermophila strain TM-1 is a moderate thermophile in the Archaea domain. It can utilize acetate, methanol and methylamines as growth substrates. In the past decades, one carbon metabolism in acetate catabolism has been well established in Ms. thermophila, but the details of the path of electron flow and energy conservation are less well understood. The carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) enzyme complex of M. thermophila is a key enzyme in acetate metabolism and previous studies showed that ferredoxin A accepts electrons from CODH/ACS. The electrons are then donated to iron-sulfur flavoprotein (Isf). Isf was partially characterized and contains iron-sulfur cluster and FMN. As a result, the properties of the Fe-S cluster and FMN were examined. Site-directed mutagenesis was performed in an effort to identify the iron-sulfur cluster ligands.

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CHAPTER 1 METHANOGENESIS I. Microbiology. Methanoarchaea. During the past three decades, the increasing interest in methane-producing microorganisms has resulted in a rapid accumulation of knowledge. The advent of 16S rRNA sequencing introduced a new classification scheme in which all forms of life could be categorized into three "primary domains"; Eucarya, Bacteria, and Archaea (42, 130). These three domains replaced the conventional classifications of either the five kingdom system or the prokaryote/eukaryote dichotomy. The Eucarya domain is comprised of plants, animals, and fungi while the Bacteria and Archaea domains contain the prokaryotes. Methane producers, extreme halophiles, sulfatereducers, and extreme thermophiles are members of the Archaea (129). All methanoarchaea belong to the Euryarcheota kingdom, and are classified into five orders, ten families, and twenty-five genera (16). The methanoarchaea represent the most diverse and extensively studied members of the Archaea domain. Despite the fact that they share the common feature of methane production, they are not closely related phylogenetically. They show diversity in: (1) morphology (rod, coccus, spirillum, and aggregate forms) (30, 57, 61, 62, 106, 108); (2) habitats with variable temperatures (2o to more than 100o C), pH (3 to 9.2), and salinity (1 mM to 3 M salt) (136); (3) cell wall components, such as pseudomurein (59), protein, glycoprotein, and heteropolysaccharides (7, 68); (4) the appearance of novel cofactors such as F430, coenzyme B, and coenzyme M in their metabolic pathways; and (5) the ability to grow on one- and two-carbon substrates. Growth Substrates. The sole means by which methanoarchaea obtain energy for growth is through methanogenesis (123). They are extremely specialized in using only a limited number of simple compounds as their growth substrates (Table 1) (136). They require a minimum reduction potential of ­ 300 mV to achieve growth (52).

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Most methanoarchaea are able to utilize H2 and CO2 as sources of energy and carbon (eq. 1, Table 1). However, several methanoarchaea including Methanosarcina thermophila strain TM1 lack this ability (134). Several methanoarchaea contain formate dehydrogenase, which allows them to use formate as a reductant (eq. 2, Table 1) (64). Methanobacterium thermoautotrophicum and some Methanosarcina sp. are able to oxidize CO for their growth (eq. 3, Table 1) (23). Acetate is a catabolic product of many fermentation processes; however, only Methanosarcina and Methanosaeta species can ferment acetate to CO2 and methane (eq. 4, Table 1) (136). The methanoarchaea in the genus Methanosarcina are able to catabolize methyl containing compounds such as methanol and methylamine (eqs. 5-7, Table 1). Utilization of short chain alcohols such as ethanol has been observed in some hydrogenotrophic methanoarchaea (eq. 8, Table 1) (128, 133). A small number of methylotrophic species utilize di-methylated sulfide (eq. 9, Table 1) (91). Ecology. Biological methane production is a strictly anaerobic process; thus, methanoarchaea are exclusively found in anaerobic environments, although some can tolerate a brief exposure to O2. Methanoarchaea can be found in diverse anaerobic habitats such as marine and freshwater sediments, hot springs, sites of geothermal activity, and in ruminant animals. They have also been found in association with human activities such as rice paddy fields, sewage sludge digesters, and landfills. Methanoarchaea are restricted to only a few substrates that in nature are provided by other microbes (136). Many such metabolic interactions among microbes in different communities can occur (109), some examples of which are: neutralism, mutualism, symbiosis, or competitive interaction. Environments containing sulfate (SO42-)-reducing microbes and methanoarchaea involve competitive interactions. Sulfate reducing microbes have been reported to grow on H2 plus CO2 and acetate; in addition, SO42reducers have a higher affinity for these substrates than methanoarchaea (97, 103). In nutrient limited environments, SO42- reducers out-compete methanoarchaea for these substrates resulting in inhibited growth of the latter (103).

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Table 1. Substrates for methanogenesis. Reactants 1) 4H2 + HCO3 + H 3) 4CO + 5H2O 4) CH3COO- + H2O 5) 4CH3OH 6) CH3OH + H2 7) 4 (CH3) 3-NH + 9H2O 8) 2CH3CH2OH + HCO39) 2(CH3)2-S + 3H2O

+ +

Products CH4 + 3H2O CH4 + 3HCO3CH4 + 3HCO3- +3 H+ CH4 + HCO33CH4 + HCO3- + H2O + H+ CH4 + 3H2O 3CH4 + HCO3- + 4NH4+ + 3H+ 2CH3COO- + CH4 + 3H2O 3CH4 + HCO3- + 2H2S + H+

2) 4HCO2- + H+ + H2O

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The methanoarchaea execute the terminal step in the degradation of complex biomass to methane (Fig 1) (40), which is very important for the global carbon cycle. The microbial degradation of biomass requires three inter-dependent metabolic groups of microbes (37). The fermentative microorganisms degrade the large complex molecules such as cellulose to the simple molecules H2, CO2, formate, and acetate, as well as various fatty acids. Then, acetogens metabolize fatty acids into H2 and CO2, formate, and acetate. Finally, methanoarchaea reduce CO2 with H2 or formate to methane, and ferment acetate to methane and CO2. Hydrogen-producing acetogens also provide substrates for methanoarchaea. The methanoarchaea in turn maintain a low H2 partial pressure that is beneficial to acetogens because high concentrations of H2 inhibit the acetogens' metabolic activity. Much of the released methane is utilized by methane oxidizing bacteria, called methylotrophs, as their growth substrate (67). However, a large amount of methane escapes and reaches the atmosphere where it is a major greenhouse gas (118). About 1% annual increase of methane in the atmosphere has been observed (136), and is mainly due to human activities. Recent work has focused on using methanoarchaea in bioremediation. In sewage sludge digesters, methanoarchaea and a mix of other anaerobic microorganisms degrade organic waste into methane which can then be used as an alternative energy source (90). In addition, studies are being performed on the ability to detoxify pollutants produced by industry and agriculture (65, 66, 100).

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Complex organics H2 + HCOOCO2 CH3COOCO2-reducing acetogenic bacteria

CH3CH2COOCH3CH2CH2COO-

Fermentative bacteria

H2-reducing acetogenic bacteria CO2 H2

Methanogenic archaea CH4 CH4 CO2 + CH4

Figure 1. Microbial food chain (40). Three different metabolic groups of microbes are required to decompose complex molecules to methane and carbon dioxide. The principle intermediates and the major route of carbon flow (solid lines) are shown.

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II. Biochemistry. Methanoarchaea are diverse in physiology and phylogeny; however, they show similarity in their metabolic pathways. These unique biochemical processes involve several novel coenzymes (Fig 2) (125). Coenzymes. Methanofuran (MF), a low molecular weight C1-intermediate carrier, has been found in methanoarchaea and a SO42- reducing archeaeon, Archaeglobus fulgidus (60, 126). MF binds CO2 and forms formyl methanofuran in the first step of the CO2 reduction pathway (76). Tetrahydromethanopterin (H4MPT) has a similar structure and function to tetrahydrofolate found in the Eucarya and Bacteria domains (123). Tetrahydrosarcinapterin (H4SPT), isolated from Methanosarcina sp., has an additional glutamyl group which differs from H4MPT (119). H4MPT has been isolated from methanoarchaea, Archaeoglobus fulgidus, and the methylotroph Methylobacterium extorquens AM1 from the Bacteria domain (19). Coenzyme M is first found in methanoarchaea and is the smallest of all coenzymes known. The structure is a thiol attached to sulfonic acid by two methylene groups (110). Coenzyme M is methylated and CH3CoM is further reduced to methane by CH3CoM methylreductase. This reaction is found in all methanogenesis pathways (29). Coenzyme B is a low molecular weight, heat stable, oxygen-sensitive compound (29). It contains a reactive thiol group which donates electrons in the methyl reductase reaction of CH3CoM (32). Factor F430, named for its characteristic absorption at 430 nm (46), is a Ni-porphyrin cofactor that is tightly associated with methylreductase (24-26, 28, 127). F430 is present exclusively in the methanoarchaea (27). F420 has structural resemblance to FMN or FAD, but it is an obligate 2-electron carrier equivalent to NAD(P) (123). The redox potential of F420 is in a range of -340 to ­350 mV. Due to the strong fluorescence of F420, it has been used to determine the presence of methanoarchaea in mixed cultures. Factor III is a corrinoidcontaining compound. It is a component of methyl transferases and the carbon monoxide dehydrogenase/acetyl CoA synthase complex (78).

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Figure 2. Structure of the coenzymes involved in methanogenesis (125).

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Methanoarchaea also contain cofactors that are commonly found in Eucarya and Bacteria such as thiamin, riboflavin, pyridoxine, biotin, niacin, panthothenate, p-amino benzoic acid, and molybdopterin (59, 75). CO2 reduction pathway. Our knowledge of methanogenesis from the CO2 reduction pathway is mostly derived from studies of Methanobacterium thermoautotrophicum strains H and Marburg (37). Figure 3 illustrates the CO2 reduction pathway. The process is conducted by several one-carbon (C1) intermediate carriers (14). Electrons for reductive steps in the pathway are derived from the oxidation of H2 by hydrogenase or formate by formate dehydrogenase (FDH) (14). Hydrogenases are classified into 3 groups based on their metal composition: NiFe-dehydrogenase (5, 9), NiFeSe-hydrogenase (86, 131), and Fe-hydrogenase (4). When cells are grown in formate, FDH oxidizes formate to CO2, which then enters the pathway (107). Molybdenum- and tungsten-containing formate dehydrogenases have been identified (1113, 101, 102). Tungsten-FDH has higher O2 sensitivity than Mo-FDH. FDH contains FAD, molybdopterin, nonheme iron and acid labile sulfur (8, 56, 116). The FDH of Methanocoocus vannielii also contains Se (58). The CO2 reduction pathway is initiated by transferring CO2 to MF followed by the reduction of CO2 to formyl-MF (76). The formyl group is then transferred to the C1carrier H4MPT to produce 5-formyl-H4MPT. Reduction of the formyl moiety proceeds via F420H2 and involves methenyl, methylene, and methyl redox states (104). Next, the methyl group is transferred to coenzyme M by a corrinoid-containing methyltransferase (115). The methyl group of CH3-CoM is finally reductively demethylated to CH4 by the enzyme complex methyl-CoM methyl reductase (MCR) (32, 33). Electrons for methyl CoM reduction are derived from coenzyme B which, after oxidation, bonds with coenzyme M to form the heterodisulfide CoM-S-S-CoB as a byproduct (34). Two MCR isoenzymes have been identified. Expression of the enzymes is growth phase dependent (88, 93) and is correlated to H2 levels in the growth medium (15, 83, 120). Recently, the crystal structure of MCR I was determined and has helped elucidate the active site and

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XH2 + MFR H2O + X

CO2

CHO-MFR

H4MPT MFR

CHO-H4MPT

H2O

CH H4MPT

F420H2 F420

CH2 H4MPT

F420H2 F420

CH3-H4MPT

CoM-SH H4MPT

CH3-S-CoM

CoB-SH CoM-S-S-CoB

CH4

Figure 3. The CO2 reduction pathway of methanogenesis (125). X, unknown electron carrier; MFR, methanofuran; H4MPT, tetrahydromethanopterin; HS-CoM, coenzyme M; HS-CoB, coenzyme B.

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catalytic mechanism (35). The MCR enzymes contain the F430-Ni porphyrin coenzyme in which Ni (I) is the catalytically active form (6, 54, 99). The CoM-S-S-CoB is regenerated to active thiol compounds by a heterodisulfide reductase (49, 51, 104). Electrons are derived from either H2 or formate. Acetate fermentation pathway. The fermentation of acetate contributes twothirds of all biologically produced methane. Figure 4 summarizes the pathway of acetate conversion to methane and CO2. In summary, the methyl group of acetate is reduced to methane by electrons derived from the oxidation of the carbonyl group to CO2. Perhaps the best characterized acetate fermentation pathway is from Methanosarcina thermophila TM1. Methanosarcina and Methanosaeta are capable of growth on acetate; however, they exhibit different affinities for acetate. At high acetate concentrations Methanosarcina predominate, whereas in acetate-limited environments Methanosaeta out-competes Methanosarcina (82, 89, 124, 135). The first step of the acetate fermentation pathway requires activation of acetate. In Methanosarcina, acetate kinase (3) and phosphotransacetylase (79) activate acetate to acetyl CoA. In Methanosaeta this reaction is catalyzed by a single enzyme, acetyl CoA synthetase (acetate thiokinase) (55). The carbon monoxide dehydrogenase/acetyl CoA synthase (CODH/ACS) enzyme complex is a central enzyme in the pathway. CODH/ACS catalyzes the cleavage of the C-C and C-S bonds of acetyl CoA, the oxidation of CO to CO2, and transfer of the methyl group to H4SPT (95, 113). CODH/ACS enzymes are widespread in procaryotes from both the Bacteria and Archaea domains, and play roles in oxidation of CO, synthesis of acetyl CoA, or cleavage of acetyl CoA (38). Enzymes from Methanosarcina sp. contain 5 different subunits which can be divided into three components (1, 43, 69). Based on the studies of CODH/ACS from M. thermophila and Clostridium thermoaceticum, the first component, a Ni/Fe-S enzyme comprised of two subunits, cleaves acetyl CoA and transfers the methyl moiety to the second component (53, 77). The Ni/Fe-S component also oxidizes CO and reduces ferredoxin (111, 112, 114). The second component, the two-subunit Co/Fe-S enzyme, contains factor III in which the active Co (I) is methylated (1, 53). The Co/Fe-S

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component is a methyltransferase that transfers the methyl group from Co (III) to H4SPT resulting in CH3-H4SPT (44, 45). The third component is very unstable and only the truncated subunit from M. barkeri has been characterized. It appears to have acetyl transferase activity that is responsible for binding of CoA and acetyl CoA (45). The genes encoding the five subunits of CODH/ACS from M. thermophila cluster in an operon (cdh) (80). In addition to the genes encoding the five subunits, a sixth open reading frame (ORF) is co-transcribed with the cdh operon. It was suggested that this ORF may encode a protein required for maturation of CODH/ACS (36, 39). The methyl moiety on CH3-H4SPT is finally transferred to CoM by methyl transferase. Methyl transferase from acetate grown cells has not been characterized. However, it is proposed that a corrinoid-containing methyl transferase, as present in CO2-reducing methanoarchaea, is likely to be involved (36, 37). CH3-SCoM is reductively demethylated to methane in the same way as in the final step in the CO2 reduction pathway. Electrons derived from CO oxidation by the Ni/Fe-S component are used to reduce ferredoxin (1, 114). Ferredoxin from M. thermophila contains 2 [4Fe-4S] centers (20), which are potentially coordinated by two cysteine motifs of CXXCXXCXXXCP (21). Electrons from ferredoxin are eventually transferred to heterodisulfide reductase (HDR) to generate the active sulfhydryl forms of CoM and CoB, as described in the CO2reducing pathway. A reconstituted CO:CoM-S-S-CoB oxidoreductase system can be established with the following purified components: ferredoxin, CODH/ACS, membranes, and HDR (92). Electron carriers between ferredoxin and HDR have been identified, including novel iron-sulfur flavoprotein (Isf) and membrane bound carriers. Heterologously-produced Isf is a homodimer containing two FMN, 7-8 Fe and acid labile S. Isf stimulates electron transfer from ferredoxin to the heterodisulfide reductase (74). Further characterization of Isf is described in chapters 4 and 5. At present, what is known about the electron transport chain is drawn from the CO:CoM-S-S-CoB oxidoreductase system. The midpoint potentials (Em) determined for each component is consistent with the electron flow as CODH ferredoxin Isf cytochrome B heterodisulfide reductase (36), but other components may be required. HDRs from M. thermophila and

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M. barkeri have been purified (72, 105), and both contain b-type hemes and 4Fe-4S clusters. They lack FAD, which is different from the HDR of M. thermoautotrophicum.

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H+

*

Membrane

+ CH3#CO2-

Ack CoA

Pta

*

CH3#COSCoA

CdhC H2O Cam Isf H + H CO

+ # 3

CdhB

#

CO2 FdxA e-

CdhA CO

*

CdhD CdhE THSPt

CH3-THSPt CH3-S-CoM

MTase

Cyt b

HS-CoM

*

e-

Hdr

HS-CoB

a Mcr

*

CH4

CoM-S-S-CoB

i Mcr

e-

FdxA

Membrane

Figure 4. Proposed pathway for acetate conversion to CO2 and CH4 in Methanosarcina thermophila. Ack, acetate kinase; Pta, phosphotransacetylase; CdhABCDE, CODH/ACS CO dehydrogensae/acetyl-CoA synthase complex; THSpt, tetrahydrosarcinapterin; FdxA, ferredoxin A; Isf, iron-sulfur flavoprotein; Cyt b, cytochrome b complex; Cam, carbonic anhydrase; MTase, methyltransferase; Mcri,methyl CoM reductase (inactive); Mcra, methyl CoM reductase (active); Hdr, heterodisulfide (CoM-S-SCoB) reductase. The carbon atoms of acetate are marked with* and # to distinguish between the carboxyl and methyl groups.

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III. Bioenergetics and electron transport carriers. As in organisms belonging to the Bacteria and Eucarya domains, ATP is a general currency in methanoarchaea. Substrate level phosphorylation and electron transport phosphorylation are the two major mechanisms for ATP synthesis in all procaryotes. So far, there is no evidence for ATP synthesis by substrate level phosphorylation in methanoarchaea (85). It has been proposed that a chemiosmotic mechanism with electron transport-driven phosphorylation is required for ATP synthesis (117). Several experiments have been performed to test this hypothesis. A number of thermodynamically favorable reactions associated with ion gradient formation have been described. Methanoarchaea use both proton and sodium gradients for ATP synthesis and endergonic reactions. The formation of ion gradients occurs during electron transfer through a membrane-bound pathway that results in reduction of CoB-S-S-CoM. This reduction is dependent on H2, F420, or ferredoxin in different methanogenic pathways (94). Knowledge of electron transfer pathways in methanogenesis is limited. Not all electron carriers involved in this process are known; however several redox cofactors from methanoarchaea have been identified, purified, and characterized. Although some of these cofactors can serve as electron carriers, their physiological roles are uncertain. Examples of known electron carriers involved in the electron transfer process are ferredoxin, cytochromes b or c, and F420. Ferredoxins are small, acidic redox proteins which contain clusters of non-heme irons and acid labile sulfides. These iron-sulfur clusters are ligated to proteins by cysteines. Clusters which serve as redox centers have been identified as [2Fe-2S], [3Fe4S], and [4Fe-4S] cluster types. The reduction potentials of ferredoxins range from ­145 to +400 mV (81), which are suitable for a variety of redox reactions (18, 132). Unlike several other iron-sulfur proteins, ferredoxin shows no enzyme activity. Ferredoxins from the methanoarchaea M. thermophila strain TM1 (20, 112, 114), M. barkeri strains MS (22, 84) and Fusaro (47), and Methanococcus thermolithotrophicus (48) have been

14

purified and characterized. These ferredoxins have been shown to be central electron carriers in anabolic and catabolic pathways in methanoarchaea (3, 4, 41, 47, 48, 111) Polyferredoxins are a class of proteins containing multiple iron-sulfur clusters. Polyferredoxin from M.thermoautotrophicum contains 12[4Fe-4S] clusters (50, 96). The genes encoding polyferredoxin (mvh B) from M. thermoautotrophicum, Methanothermus fervidus, and Methanococcus voltae are located in the methyl viologen (MV) hydrogenase (mvh) operon and are conserved (50). Therefore, polyferredoxin has been proposed to function in association with the MV-hydrogenase system; however, the reduction of polyferredoxin by MV-hydrogenase in the presence of H2 is very low. The second possible role for polyferredoxin is iron-storage (50, 96). Cytochromes are ubiquitous membrane-bound electron transfer components in nature. Only the methanoarchaea that are able to grow on methyl-containing compounds, such as acetate, methanol and methylamine, contain cytochromes (71). This indicates that cytochromes are not involved in methane formation from CO2 and H2, or formate. It has been proposed that cytochromes function in the methyl oxidation of these methylcontaining compounds (71). Two b-type cytochromes and one c-type cytochrome were detected in methanol- and methylamine-grown cells whereas the acetate-grown cells contain an additional b-type cytochrome (70). The midpoint potentials for b-type cytochromes found in methanol-, methylamine- and acetate-grown cells are ­325, -183, and ­253 mV, respectively (70). Recently, the membrane fraction from methanol-grown Methanosarcina mazei Gö1 revealed two b-type cytochromes and two c-type cytochromes (63). The midpoint potentials for the b-type cytochromes are ­135 and -240 mV and -140 and -230 mV for the c-type cytochromes (63). There has been evidence suggesting that cytochromes are involved in electron transfer between F420H2 and CoMS-S-CoB (63). F420 is another required electron carrier for methanogenesis. It is an obligate twoelectron carrier in Archaea which functions in analogy to NAD and NADP in the Eucarya and Bacteria domains. The variable amounts of F420 among methanoarchaea may be due to different requirements for this electron carrier in diverse metabolism (31).

15

Proteins known to interact with F420 include F420-reducing hydrogenase, NADP-F420 oxidoreductase, formate dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, secondary alcohol dehydrogenase, puruvate synthase, and -ketoglutarate synthase (29). The FMN-containing flavoprotein, flavoprotein A, was recently purified, cloned, and sequenced from M. thermoautotrophicum strain H and Marburg (87, 121). It copurified with the H2:heterodisulfide oxidoreductase complex. The flavoprotein A from strain Marburg was purified as a homotetramer with a 43 kDa molecular mass per subunit whereas the one from strain H was a homodimer with a monomeric molecular mass of 45 kDa. The expression of either flavoprotein increased when cells were grown in iron depleted media. The physiological role for flavoprotein A remains speculative, although the FMN containing property suggests a function as an electron carrier. It may function to substitute an essential iron-containing protein during iron starvation. Polyferredoxin seems to be the most feasible protein for which flavoprotein A can substitute. With many completed genome sequencing projects, a number of flavoprotein A related protein sequences have been identified. The sequence comparisons reveal a conserved region for FMN binding (122). Methanophenazine is a recently discovered redox-active cofactor. It was first isolated from the membranes of M. mazei Gö1 and shown to be very hydrophobic. The structure is a 2-hydroxy phenazine derivative connected to a polyisoprenoid by an ether bond. It has a molecular mass of 538 Da (2). The 2-hydroxy phenazine (a soluble analogue of methanophenazine) is able to accept electrons from both F420-hydrogenase and MV hydrogenase (2). Furthermore, reduced methanophenazine donates electrons to heterodisulfide reductase from M. thermophila (10). Therefore, methanophenazine has been proposed to play an important role in vivo in membrane-bound electron transport systems of F420-hydrogenase: heterodisulfide oxidoreducatase and H2: heterodisulfide oxidoreducatase. Diphenyleneiodonium chloride (DPI), a competitive inhibitor of methanophenazine, inhibited both membrane-bound electron transport systems of M. mazei Gö1 (17).

16

Preliminary investigation of M. thermoautotrophicum membranes is consistent with the presence of low potential electron carriers (73). EPR studies suggest that ironsulfur clusters are membrane components. Upon the addition of either F420 or CH3-CoM to membranes, the EPR spectra changes, results which are consistent with the involvement of membrane components in the electron transfer process (98).

17

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Robinson, J. A., and J. M. Tiedje. 1984. Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions. Arch. Microbiol. 137:26-32.

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Rogers, K. R., K. Gillies, and J. R. Lancaster Jr. 1988. Iron-sulfur centers involved in methanogenic electron transfer in Methanobacterium thermoautotrophicum. Biochem. Biophys. Res. Commun. 153:87-95.

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Schink, B. 1988. Principles and limits of anaerobic degradation: environmental and technical aspects. Biology of anaerobic bacteria. Zehnder A.J.B. ed. Wiley Interscience , New York. 771-846.

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Schmitz, R. A., S. P. J. Albracht, and R. K. Thauer. 1992. A molybdenum and a tungsten isoenzyme of formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei. Eur J Biochem. 209:1013-1018.

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Schmitz, R. A., S. P. J. Albracht, and R. K. Thauer, 1992. Properties of the tungsten-substituted molybdenum formylmethanofuran dehydrogenase from Methanobacterium wolfei. FEBS Lett. 11479:78-81.

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Schonheit, P. J., K. Kristjansson, and R. K. Thauer. 1982. Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate. Arch. Microbiol. 132:285-288.

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Schworer, B., and R. K. Thauer. 1991. Activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase in methanogenic bacteria. Arch. Microbiol. 155:459-465.

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Simianu, M., E. Murakami, J. M. Brewer, and S.W. Ragsdale. 1998. Purification and properties of the heme and iron-sulfur containing heterodisulfide reductase from Methanosarcina thermophila. Biochemistry. 37:10027-39.

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CHAPTER 2 IRON-SULFUR PROTEINS I. Introduction. Since their discovery in the 1960s, a vast amount of data has been accumulated regarding iron-sulfur proteins. These proteins are abundantly found in nature and are regarded to be an ancient component of a chemoautotrophic process in which a Ni/Fe-S center catalyzes carbon fixation (39). Although most iron-sulfur proteins are electron carriers, they have also been shown to possess other functions such as providing substrate binding sites, general structural roles, iron storage, and gene regulation. Developments in the fields of biophysical techniques, molecular biology, chemical synthesis, and computer modeling, in conjunction with site-directed mutagenesis methods, have resulted in a rapid accumulation of data concerning iron-sulfur proteins. Insight regarding cluster assembly, protein stability, electronic structure, functions, and ligand coordination has been revealed. II. Structure and properties of clusters. Iron-sulfur proteins are broadly classified as either simple or complex. The first group contains only iron-sulfur clusters, whereas the latter contains additional prosthetic groups (42). Clusters in iron-sulfur proteins, rather than single-metal sites, provide the diversity and versatility for these proteins to function properly (76). The clusters in ironsulfur proteins contain Fe with at least partial S coordination (42). Their structures are Fe2+ or Fe3+ with approximately tetrahedral coordination to S atoms of cysteine residues and inorganic sulfides. Inorganic sulfides are sometime referred to as "acid-labile sulfides", because the sulfur atoms have a ­2 valence state that are released as H2S with acid treatment (85). Cysteine appears to be a major residue involved in cluster bridging to the polypeptide; nonetheless, evidence of non-cysteinyl ligands has recently emerged. The structure/function basis for non-cysteinyl versus cysteinyl ligands has not been determined; however, it appears that when Fe is coordinated with non-cysteinyl ligands the cluster is involved in substrate binding and there is a shift in reduction potential (42).

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The properties of iron-sulfur clusters are dependent upon their electronic configuration and capability of electron delocalization. Iron-sulfur proteins show a very broad range of midpoint potentials. This can result from several factors including nature of cluster ligands, hydrophobicity and charge of residues in the surrounding polypeptide environment (61, 82). Electron paramagnetic resonance (EPR) and Mössbauer spectroscopies are the techniques widely used to examine iron-sulfur clusters in proteins and their properties. EPR spectroscopy is applicable to paramagnetic systems, and it is a sensitive method which is ideal for studying many metalloproteins. Information obtained from EPR analysis included electronic structure, metal coordination-sphere composition and geometry (52). On the other hand, Mössbauer spectroscopy gives detailed pictures of the chemical state of the iron atoms as well as the electron distribution in various redox states of different iron-sulfur cluster types. Information derived from Mössbauer spectra include the nature and arrangement of the ligand, spin of the iron atoms, spin coupling, and the surrounding protein environment (41). To obtain an adequate Mössbauer spectrum, the sample must contain a certain minimum quantity of the Mössbauer isotope, which is 57Fe for iron-sulfur proteins. Due to the low natural abundance of 57Fe, enrichment of the sample with 57Fe is always necessary (41). [1Fe] cluster type. Basic structures of Fe-S clusters and their properties are shown in Figure 1 (42). This cluster type, also referred to as rubredoxin-type cluster, contains a single Fe atom bridged with four cysteines. The proteins show intense red color when oxidized due to the charge transfer of S Fe (17, 53). The structures of rubredoxins from several microbial species have been solved. The EPR spectrum of the ferric protein indicates the presence of high spin iron (S = 5/2) with g values of 4.3 and 9 (66). The oxidized rubredoxin with high spin Fe3+exhibits a small chemical shift at 0.25 mm/s at 77 oK and three quadrupole doublets in the Mössbauer spectrum (68, 71). A large quadrupole split (3.16 mm/s at 77 oK) and small chemical shift (0.65 mm/s at 77 oK has been observed for the Fe2+ form (68, 71). The coordinating cysteine motifs, CXXC, at both the N- and C- terminal are conserved among these proteins (65). The midpoint potential of the cluster is in the range of +20 to ­60 mV.

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[2Fe-2S] cluster type. This cluster type is composed of a Fe2S2 (S-cys)4 unit. The oxidized form ([2Fe-2S]2+) contains two Fe3+ ions. When reduced by one electron, a mixed valence of Fe3+- Fe2+ ([2Fe-2S]1+ cluster results. This type of cluster is always described as a "plant-type ferredoxin", since it is present in photosynthetic organisms. However, this cluster type has also been characterized from ferredoxins of halophiles, and species in the genera Rhodobacter, Clostridium, and Azotobacter from the Bacteria domain. (31). A [2Fe-2S] subclass called the Rieskie cluster describes iron-sulfur proteins where the [2Fe-2S] clusters are coordinated by non-cysteinyl ligation from two histidines (55). This subclass has a higher reduction potential than other [2Fe-2S] proteins with exclusively cysteine ligands. The sequence motif for this subclass is CXHX15-17CXXH (7, 15). All of the sequences known for coordination are shown in Figure 2. The two Fe3+ with S = 5/2 are antiferromagnetically coupled in the oxidized state and, thus, are EPR silent. Conversely, with the reduced state of [2Fe-2S]+, the antiferromagnetic couple results in S = _, and the typical gav value derived from EPR is 1.96 for plant type ferredoxin and 1.90 for Rieskie proteins (81). The Mössbauer spectrum for the oxidized proteins shows a quadrupole split with a small chemical shift value, while the reduced proteins give two quadrpole doublets. One doublet corresponds to the Fe3+ ion in the cluster, and the other with larger quadrupole is derived from Fe2+ ion in the cluster (41). [3Fe-4S] cluster type. This cluster type was first interpreted as an artifact of a degradative product of a [4Fe-4S] center (6), because [4Fe-4S] centers can be interconverted to [3Fe-4S] centers by removal of an iron unit. However, a considerable amount of data has shown that the [3Fe-4S] cluster is indeed a true cluster in nature for some proteins. The core structure is Fe3S4 (S-cys)3 which forms a cube-like structure due to a missing Fe. The [3Fe-4S] center involves one electron transfers with 1+ and 0 reduction states. In the 1+ oxidation state, all Fe3+ are antiferromagnetically coupled which results in S = _. The EPR signal for this [3Fe-4S]1+ cluster is either axial or rhombic with the resonance at gav values of 2.02. The Mössbauer spectrum of oxidized ferredoxin II from Desulfovibrio gigas is a single quadrupole doublet with chemical shift at 0.27 mm/s characteristic of the Fe3+ in tetrahedral S coordination. The reduced cluster

34

produces a spectrum with two different intensities (2:1) of quadrupole doublet (40). The less intense signal derived from Fe3+ is unchanged upon reduction, as indicated by the Mössbauer parameter (40). Several sequence motifs coordinating [3Fe-4S] are shown in Figure 2. The motifs contain two cysteines located closely to each other and another distal cysteine. The cysteine motif coordinating the [4Fe-4S] cluster that is easily converted to [3Fe-4S] cluster mostly contains aspartate in the place of the second cysteine (9, 25, 42). [4Fe-4S] cluster type. This cluster has a Fe4S4 (S-cys)4 cubane structure. It is involved in one electron transfer reactions with three associated oxidation states, +3, +2, and +1 (10, 11, 34). A cluster that undergoes three oxidation states in a biological system has not been reported. These cluster types have a wide reduction potential in the range of +450 to ­700 mV. The [4Fe-4S] cluster with low reduction potential stays in the +2/+1 state, while the high reduction potential proteins (HIPIPs) stay in the +3/+2 state (31). The low reduction potential of reduced [4Fe-4S]1+ and the oxidized HIPIPs with [4Fe4S]3+ are EPR active species. EPR analysis for [4Fe-4S]3+ of HIPIPs yields an axial signal with g = 2.2 and 2.04 whereas the [4Fe-4S]1+ exhibits a rhombic type EPR with g = 20.6, 1.92, 1.88 with gav < 2. Mössbauer spectra of [4Fe-4S]2+ centers are generally broad and also indicate almost equivalent iron atoms in the cluster. When the [4Fe-4S]2+ is oxidized or reduced, the spectra indicate an inequivalence of iron atoms in the clusters. This feature is clearly evident from the spectra obtained in an applied magnetic field (41).

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Cluster type

oxidation and spin number Fe2+ S = 2 Fe3+ S = 5/2 [2Fe-2S]+ S = _ [2Fe-2S]2+ S = 0 [3Fe-4S]0 S =2 [3Fe-4S]+ S =1/2

EPR (g)

Mössbauer ( ) 0.65 0.25 0.25, 0.55 0.26 0.30, 0.46 0.27

4.3, 9 1.89, 1.96, 2.05 1.97, 2.00, 2.02

[4Fe-4S]+ S = 1/2 or 3/2 [4Fe-4S]2+ S = 0 [4Fe-4S]3+ S = _

1.88, 1.92, 2.06 2.04, 2.04, 2.12

0.57 0.42 0.31

Figure 1. Structures, and properties of the structurally characterized iron-sulfur centers that are involved in biological systems (42, 52).

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A. N----CX4CX2CX29C----C N----CX5CX2CX36/37C----C N----CX3CX2CX36C30/31CX59C----C N----C-X2CX9CX31CX3C----C N----CXHX15-17CX2H----C B. N----CX6C----C (subunit 1) N----CX6C----C (subunit 2) [4Fe-4S] Fx in photosystem I N----CX34C----C (subunit 1) N----CX34C----C (subunit 2) N----C---------CX2C---------C N----CX2CX16CX13C----C N----CX6CX2CX5C----C N----CXCX20CX20C----C N----CX2CX11CX5C----C N----CX2CX2C---------C-P----C N----CX2CX2CX3C-P-------CX2CX2-8CX3CP----C C. N----CX5C---------CP----C N----CX2DX2C---------CP----C [3Fe-4S] ferredoxins [3Fe-4S] or [4Fe-4S] ferredoxins D. N----CX2DX2CX3CP-------CX2CX2CX3CP----C N----CX7CX3CP--------- CX2CX2CX3CP----C 2[4Fe-4S] or [3Fe-4S] plus [4Fe-4S] ferredoxin [4Fe-4S][3Fe-4S] ferredoxins [4Fe-4S] nitrogenase [4Fe-4S] aconitase [4Fe-4S] HIPIP [4Fe-4S] Endonuclease [4Fe-4S] Leucine rich repeats [4Fe-4S] Leucine rich variants [4Fe-4S] ferredoxins 2[4Fe-4S] ferredoxins Plant-type ferredoxins Hydroxylase-type ferredoxins Biotin synthases Clostridium pasteurianum ferredoxin Rieskie proteins

37

Figure 2. Arrangement of residues involved in coordination of [2Fe-4S] (A), [4Fe-4S] or 2[4Fe-4S] (B), [3Fe-4S] or [4Fe-4S] (C), and 2[4Fe-4S] or [3Fe-4S] plus [4Fe-4S] (D) clusters. "N" and "C" at the beginning and the end the motifs represent the N and C terminal sequence of the proteins. Dashed lines indicate variable spacing between residues. C, D, and H within the motif are cysteine, aspartate, and histidine respectively. Underlined and bolded residues are those considered to be ligands in each cluster based on X-ray structure, sequence homology, site directed mutagenesis, or spectroscopic evidence (21, 42).

38

III. Ligation of iron-sulfur clusters Cysteine is the major ligand in nature that coordinates iron atoms in iron-sulfur clusters (62). Nonetheless, several different ligands, such as aspartate, histidine, or water, have been identified. Direct information from crystal structures as well as through other various spectroscopic studies has helped to identify the ligands involved in iron-sulfur cluster coordination. Aconitase is an enzyme containing a 4Fe-4S center that is not involved in redox chemistry, but rather it is a substrate binding site (4, 49). It is an enzyme that catalyzes the interconversion of citrate and isocitrate in the TCA cycle. The enzyme has been characterized by different approaches. The crystallographic structures of the enzyme alone, with substrates, and inhibitors have been determined. In the absence of substrate, the active 4Fe-4S cluster is coordinated by 3 cysteines of the protein and an oxygen atom from solvent is the fourth ligand (4, 74). In the presence of substrate, the crystal structure shows a 4Fe-4S center in agreement with previous electron nuclear double resonance (ENDOR) results. Both methods show that one Fe in the cluster has octahedral geometry rather than tetrahedral. This octahedral geometry is due to additional ligands from the carboxy and hydroxy groups of substrate and one water molecule (4, 49, 50). The hydrogenase from Desulfovibrio gigas is another example of a non-cysteinyl coordination that is shown by crystal structure. This protein contains a 4Fe-4S cluster and Ni as prosthetic groups. The structure revealed the presence of three cysteines and one histidine ligand to the 4Fe-4S center (83). Albeit that the crystal structure is always a useful tool to determine ligand identity, it is not easy to obtain the crystal structure for all proteins. Hence, other methods often must be used. Iron-sulfur proteins are suspected of having non-cysteinyl ligation if they have similar spectroscopic and catalytic characteristics to known noncysteinyl coordinated iron-sulfur proteins. Mostly, sequence comparisons with homologues in combination with spectroscopic and site-directed mutagenesis studies have been used to help identify the ligands for iron-sulfur clusters in proteins. For

39

example, electron spin echo envelope modulation (ESEEM) and ENDOR spectroscopy indicate that Rieskie proteins contain 2Fe-2S centers that are coordinated by two cysteines and two histidines (7, 18, 26, 27). Histidines and cysteines are conserved among Rieskie type proteins. In addition, they exhibit higher reduction potentials than a 2Fe-2S cluster coordinating solely cysteines (55, 81). Another mixed ligand coordination has been observed in [4Fe-4S] ferredoxins from sulfate reducers and hyperthermophilic archaea. Ligands to the iron-sulfur clusters in these proteins are cysteines and aspartate. The [4Fe-4S] binding motif in this group is similar to the conventional cysteine motif (CX2CX2C and a distal C) except the second cysteine is substituted by aspartate (8, 32, 64). These ferredoxins exhibit one similar property which is the 3Fe-4S/4Fe-4S interconversion. Even though Pyrococcus furiosus ferredoxin contains a [4Fe-4S] center, it exhibits anomalous spectroscopic properties from clusters ligating with cysteine exclusively (9, 13). A number of site-directed mutagenesis studies have contributed to an understanding of iron-sulfur cluster ligation (62). Site directed mutagenesis provides not only identification of the ligand residue, it also provides an understanding of the protein environment which has a direct effect upon the iron-sulfur cluster structure and properties. Replacement of one of the ligands can result in the variant protein being either improperly folded or a variant with no iron-sulfur clusters. The conclusion from this kind of result is almost always that the replacement amino acid is involved in cluster coordination and that it also influences the protein folding (62, 79). Sometimes the variant has similar properties as wild type and, as such, this result excludes the replaced residue as a ligand to the iron-sulfur cluster. In several incidences, the residue replacing cysteine serves as a ligand in which case the variants can contain iron-sulfur clusters with altered electronic, redox potential and functional properties. The term "ligand exchange" is applied where a new residue substitutes for the missing ligand. One form of ligand exchange is the use of serine and aspartate in place of a cysteinyl ligand. Examples of this are [4Fe-4S] center variants of PsaC and [2Fe-2S] center variants of ferredoxin from C. pasteurianum (20, 43, 57). Another form of ligand exchange is the use of a free

40

cysteine from a different position to serve as a ligand rather than using the replacement residue (23, 24, 79). This type of ligand exchange is referred to as "ligand swapping" (23, 24). Ligand swapping often is associated with re-arrangement of the protein conformation (23, 24, 79). The phenomenon called "cluster conversion" occurs when the original iron-sulfur cluster is converted into another structural type. Cluster conversions can result from exposure to air, chemical oxidation-reduction, change of pH, and site-directed replacement of ligands (42). A common form of cluster conversion occurs when a cysteine ligand in a [4Fe-4S] center is replaced converting it to a [3Fe-4S] center. Additionally, a [3Fe-4S] cluster may be converted into a [4Fe-4S] cluster by replacing a noncysteinyl residue with cysteine that preserves the common cysteine motif (CX2CX2C and a distal C) for coordination of a [4Fe-4S] center (1, 8, 44, 54). Another example of cluster conversion is Clostridium pateurianum rubredoxin, in which the 1Fe cluster is changed to a [2Fe-4S]-plant type cluster by replacing a cysteinyl ligand with alanine (58). This result may be due to the structure around the metal site in rubredoxin which is similar to the [2Fe-2S] Rieske protein. Another possibility is that the variant has a large structural rearrangement allowing the additional incorporation of Fe and sulfides. IV. Function of iron-sulfur centers. A. Electron transfers. Iron-sulfur clusters found in all domains of life are capable of serving several functions. Most are electron carriers that accept, donate, and shift electrons (3). Examples are ferredoxins and HIPIPs. Ferredoxins are well-studied proteins and their structures from different organisms have been solved. The midpoint potentials of these proteins are broadly varied from positive to negative values. This property allows them to be either an electron donor or acceptor in various metabolic reactions. Examples of enzymes and proteins that ferredoxins catalyze electron transfers are hydrogenase, nitrogenase, cytochromes, nitrite reductase, nitrate reductase, sulfite reductase, pyruvate oxidoreductase, and formate dehydrogenase. HIPIPs have a broad range of positive

41

midpoint potential values, which have been suggested to be correlated to the overall charges of the polypeptide environment (30). HIPIPs are electron carriers between the photoreaction center and the cytochrome bc1 complex in phototrophic microbes from Bacteria domain (36, 37, 78). They also have been suggested to transfer electrons in thiosulfate oxidation and iron oxidation (31). B. Catalysis. Many enzymes use iron-sulfur clusters as their substrate binding and activation sites. For example, enzymes that perform dehydration and hydration activities in which iron-sulfur clusters act as a Lewis acid in the reaction (4, 42). Aconitase is the best characterized enzyme among this group. The catalytic Fe of the [4Fe-4S] center in aconitase shows no redox changes during the reaction (2). The catalytic role of iron-sulfur clusters which undergo redox changes is exemplified in the radical based mechanism reactions. Examples are anaerobic ribonucleotide reductase (RR) and biotin synthase (63). RR is involved in the first step in DNA synthesis, ribonucleotide reduction to 2'-deoxyribonucleotide. Escherichia coli uses specific RRs during aerobic and anaerobic growth conditions, both of which generate radicals required for the reaction mechanism (73). The aerobic RR is a heterodimer in which the smaller subunits contain µ-oxo diiron centers. The metal center is used to oxidize a tyrosyl residue to generate a tyrosyl radical. The anaerobic RR however, contains a [4Fe-4S] center at the interface of the two small subunits. This is the third example in which an iron-sulfur cluster is located at the interface of two polypeptides other than nitrogenase and Fx in photosystem I. The 4Fe-4S center of the anaerobic RR reduces S-adenosyl methionine (SAM) to give a 5'-deoxyadenosyl radical that generates a glycyl radical. Biotin synthase catalyses the last step of the biotin synthesis pathway (77), in which a sulfur atom in the form of a thiol derivative is inserted into dethiobiotin (16). The aerobically purified enzyme contains a [2Fe-2S] which is proposed to be involved in the generation of the 5'-deoxy adenosyl radical. Recent absorption, variable temperature magnetic circular dichroism (VTMCD), and EPR

42

spectroscopy indicated conversion of the [2Fe-2S] centers to a [4Fe-4S] center under anaerobic conditions (16). The [4Fe-4S] center form of biotin synthase is postulated to be involved in the radical generating reaction rather than the [2Fe-2S] cluster form. The physiological significant of the cluster conversion may be a tool to regulate enzyme activity due to oxidative stress (16). However, more experiments are needed to verify this hypothesis. C. Regulatory role Iron-sulfur clusters also serve a regulatory role, in which they sense molecular iron, oxygen, superoxide ion, and possibly nitric oxide concentrations (5, 22, 33, 35, 75). For example, iron responsive binding protein (IRP) regulates the iron level in higher organisms by controlling the gene expression of both iron storage ferritin and the transferrin receptor (75). IRP recognizes a region called iron responsive element (IRE) located at the 5' and 3' regions on the mRNA encoding ferritin and transferrin respectively. When the cellular iron level is low, functional IRP binds IRE on both ferritin and transferrin mRNA. This results in blocking ferritin translation and increasing transferrin mRNA stability resulting in an increased production of transferrin (46, 56). IRP is an isoform of aconitase, but contains no iron-sulfur clusters (28, 45). When iron is limiting, IRP (apo-aconitase) increases. Conversely, when iron is abundant, holoaconitase with aconitase activity is increased while IRP activity is decreased. This switching in activity of a bifunctional protein between enzyme catalysis (aconitase) and RNA binding activity (IRP) occurs by assembly and disassembly of iron-sulfur clusters which depends on the cellular iron level (3). Another example of an iron-sulfur protein serving a regulatory role is fumarate nitrate reductase regulatory protein (FNR), an oxygen sensor protein. FNR is a transcription factor, which activates a set of genes under anaerobic conditions (80). The functional enzyme form contains a [4Fe-4S] center. In the presence of oxygen, the cluster is destroyed rapidly and FNR subsequently loses its DNA binding ability. Hence, the regulatory function of FNR is controlled by the oxygen level in cells (76).

43

There is also a group of proteins with tandem repeats of leucine and aliphatic residues in their sequence called leucine-rich repeats (LRRs) and leucine-rich variants (LRVs). The X-ray crystal structure for an LRV was recently obtained. The structure shows a 4Fe-4S cluster with a distinctive motif of CXXCX11CX5C located in a small domain (67). This cysteine motif is different from the one described for the motif in LRRs (CXCX20CX20C). The [4Fe-4S] centers of these LRRs and LRVs are highly susceptible to oxygen (67), hence it is postulated that those proteins may function as an oxygen sensor; however, more evidence is needed to support this hypothesis. Sox R is another protein where the iron-sulfur cluster is proposed to serve a regulatory role. The Sox R system is a mechanism developed to protect cells from superoxide and nitric oxide. The mechanism is unknown at this time; however, the ironsulfur center seems to have a role in sensing these compounds (35, 84). D. Iron storage role. Polyferredoxin from Methanobacterium thermoautotrophicum contains 12[4Fe4S], and is thought to function as an iron storage protein or as an electron carrier (29, 72). The postulated physiological role for polyferredoxin as an electron acceptor of the MVreducing hydrogenase is obscured, since the specific rate of reduction is very slow. E. Structural role. Another role proposed for iron-sulfur clusters involves maintaining protein structure (42). The [4Fe-4S] centers in endonuclease III and Mut Y, members of the base excision repair enzyme superfamily (47, 48, 59, 60), have no catalytic role and are resistance to oxidation and reduction (14, 19). However, the enzyme activity is dependent upon the presence of these iron-sulfur clusters. These observations suggest the iron-sulfur cluster may be required for structural integrity. Although the [4Fe-4S] centers of these enzymes are not directly involved in substrate binding or catalysis, they appear

44

to be required for efficient specific DNA binding. The iron-sulfur cluster may juxtapose the catalytic or substrate binding sites to interact with DNA (69, 70). Another striking feature for this superfamily of enzymes is that they contain a similar cysteine motif of CX6CX2CX5C, which coordinates [4Fe-4S] centers (47, 48). This motif is one of the most compact cysteine motifs known to date. V. Iron-sulfur cluster assembly in proteins. Understanding the in vivo formation of iron-sulfur clusters still falls behind, in spite of the deep understanding of iron-sulfur cluster structure, reactivity, physiological roles, and properties. Two scenarios for iron-sulfur cluster synthesis have been proposed. The first is called spontaneous self-assembly. In the mechanism, iron-sulfur clusters are thought to form spontaneously in the presence of ferric or ferrous salt in aqueous mercaptoethanol with sodium sulfide (38). The logic for this process is that nature may have synthesized iron-sulfur clusters by using the available chemistry of the geosphere during evolution of the biosphere (52). This spontaneous process has been successful in replicating known metalloprotein core clusters. The other proposed scenario, assisted assembly, involves other protein components for cluster formation and insertion into the proteins. The best characterized are the Nif S, U, and M system from Azotobacter vinelandii. Nif S is a cysteine desulfurase that converts L-cysteine to L-alanine and an activated sulfur (cysteinyl persulfide). The enzyme contains pyridoxal phosphate, which is essential for catalysis (87). Nif S is proposed to be a sulfur donor in the formation of the iron-sulfur cluster core structure (87). The reconstitution of iron-sulfur clusters of apoproteins is facilitated in the presence of Nif S (12, 86). The nif S gene is highly homologous and present in different organisms, suggesting that it is a universal sulfur mobilizing agent (51, 87). Nif U and Nif M are less well characterized, but they are hypothesized to be involved in cluster formation.

45

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CHAPTER 3 OBJECTIVES Methanoarchaea obtain energy for growth by coupling electron transport phosphorylation to methane formation. An undeveloped area of research has been the identification of electron carriers. The iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila has been proposed to be an electron carrier in the acetate fermentation pathway. The main objectives of my research were to characterize the properties of the iron-sulfur center and FMN in Isf, identify the ligands required for coordination of the iron-sulfur cluster, and to identify the electron acceptor of Isf. Isf was previously shown to contain one FMN and iron-sulfur center per subunit; however, the properties of these redox centers were not characterized. Various proteins posses different iron-sulfur cluster types, each showing distinct properties. Therefore, the identity of the iron-sulfur center in Isf is important for understanding how Isf functions as a redox protein. Most iron-sulfur clusters are ligated to the polypeptide backbone primarily via cysteine residues. Several amino acid motifs for iron-sulfur cluster ligation have been reported (refer to Chapter 2). These motifs can be used as a basis for the preliminary prediction of ligands that bind to iron-sulfur cluster types in proteins. Most iron-sulfur clusters are coordinated by four cysteine residues. Sequence comparisons among Isf homologues reveal a novel motif with perfectly conserved cysteines which could possibly coordinate the iron-sulfur center in Isf (refer to Chapter 4). This putative motif is the most compact cysteine motif known for coordinating a redox active protein. The discovery of Isf as an electron carrier in the acetate fermentation for M. thermophila allows the possibility for a comprehensive study of the electron transport components in this methanoarchaeon. Determination of the midpoint potential of the iron-sulfur center and FMN will be useful in investigating the possibility of intra- and inter-molecular electron transfer processes in the acetate fermentation pathway. The

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presence of both a one-electron carrier and a two-electron carrier in the same protein suggests a one/two electron switch function for Isf. The fact that Isf has a defined redox potential and a possible one-two electron switch function provides a basis to identify the electron acceptor for Isf.

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CHAPTER 4 ELECTROCHEMICAL AND SPECTROSCOPIC PROPERTIES OF THE IRON-SULFUR FLAVOPROTEIN FROM Methanosarcina thermophila

ABSTRACT

Based on spectroscopic analyses, a heterologously produced iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila contains two [4Fe-4S] centers and two flavin mononucleotide (FMN) cofactors in the homodimeric protein. The midpoint potentials (Em) for the [4Fe-4S]2+/1+ and FMN/FMNH2 are ­ 394 and ­277 mV respectively. Interestingly, the semiquinone form of FMN was not detected during the potentiometric titration; however, a small amount of red semiquinone was found in frozen reaction mixtures of CODH/ACS and Isf. The EPR spectrum of the reduced protein showed g values characteristic of [4Fe-4S] center with additional g values (2.06, 1.93, 1.86 and 1.82) due to microheterogeneity among Isf molecules. A variety of physiological 2-electron acceptors were examined for the ability to oxidize Isf, but none are able to carry out this function. Extract from H2/CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either H2 or CO-dependent reduction of M. thermophila Isf. Furthermore, the genomic sequences of M. thermoautotrophicum and Methanocoocus jannaschii also contain Isf homologues. These results may suggest a general role for Isf as an electron carrier in both acetate fermenting and CO2-reducing methanoarchaea.

INTRODUCTION

Methane production resulting from the energy-yielding metabolism of the methanoarchaea represents the final step in the anaerobic degradation of complex materials (7). This process is unique and involves many enzymes and coenzymes found only in methanoarchaea (26). Methanoarchaea produce methane by two major pathways (7). The first is the CO2-reducing pathway in which CO2 is reduced to methane using electrons derived from either H2 or formate (equations 1 and 2). Another pathway is

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acetate fermentation, which contributes two-thirds of all biological methane produced in nature (equation 3). In this pathway, acetate is cleaved in a CoA-dependent reaction and the methyl group is reduced to methane using electrons derived from oxidation of the carbonyl group to carbon dioxide (8). CO2 + 4H2 4 HCOO- + 4H+ CH3COO- + H+ à à à CH4 + 2H2O CH4 + 3 CO2 + 2 H2O CH4 + CO2 (1) (2) (3)

Although the biochemistry and enzymology of carbon flow in these pathways are areas of intense research, the electron transport carriers in methanogenesis are poorly understood.

Recently, a homodimeric iron-sulfur flavoprotein (Isf) was identified from an acetate-utilizing methanoarchaeon, Methanosarcina thermophila. In the genome of M. thermophila, the isf gene is located upstream and transcribed in the opposite direction from the pta-ack operon encoding phosphotransacetylase and acetate kinase (14). Comparison of the deduced Isf sequence with sequences in the available databases suggested Isf was a novel iron-sulfur flavoprotein. Isf was hyperproduced in Escherichia coli. The heterologously produced protein was partially characterized and in vitro reconstitution experiments suggested Isf is a component in the electron transport chain for methanogenesis from acetate (14). The absorption spectra and chemical analyses suggested one iron-sulfur cluster and one FMN per subunit.

The present study reports the characterization of this iron-sulfur cluster and unique FMN properties to offer insight into the role of Isf in electron transport. Additionally, the context of Isf function in M. thermophila electron transport was examined by testing several known electron carriers for interactions with Isf. Lastly, information about the Isf homologs in other CO2-reducing methanoarchaea will be discussed to help understand the physiological role of this electron carrier.

Portions of data contained in this section were obtained by Dr. S.W. Ragsdale and Dr. D.F. Becker at the University of Nebraska, and Dr. K.K. Surerus at the University of Wisconsin Milwaukee. Their contributions are noted in the appropriate sections.

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MATERIALS AND METHODS

Isf sequence analysis. In the process of doing site-directed mutagenesis for ligand identification, it was discovered that the sequence at the C-terminal was incorrect from that previously reported (14) and therefore, the gene was re-sequenced. The coding region of isf was amplified using the Polymerase Chain Reaction (PCR) from Methansarcina thermophila genomic DNA. The sequence of the upstream primer was 5'GGTGCACATATGAAAATAACAGGAAT-3' and contained the recognition sequence for NdeI. The sequence of the downstream primer was 5'CAACTGGATCCATGCGATCATAAAC-3' and contained the recognition sequence for BamHI. The PCR product was restricted with NdeI and BamHI. The resulting DNA was cloned into the BamHI and NdeI sites of the pT7-7 overexpression vector. The construct was checked by sequencing using the automated dideoxy method at the Penn State University nucleic acid facility. The -EMBL3 (M. thermophila genomic library containing isf , (14)) constructs were also subjected to the same procedure as described above. Lastly, plasmid pML701 (14) was sequenced to confirm this error.

Protein production and purification. All proteins were purified anaerobically. M. thermophila Isf was overproduced in Escherichia coli BL21(DE3) and purified as described (14). M. thermophila ferredoxin A was purified as described (25). The following procedure was used to obtain 57Fe-enriched Isf for Mössbauer study. The

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Fe-labeled Isf was produced by growing E. coli cells in defined medium supplemented

with 57Fe at the final concentration of 20 µM. A solution of 57Fe was obtained by dissolved 42.5 mg 57Fe in 850 µl of 2 N H2SO4 in a tube capped with a rubber stopper that has a small plastic tubing insert at the top to let the gas out during the reaction. The reaction solution was heat at 60-65o C for a week. The iron concentration was determined by ferrozine using standard Fe(II) solution as a standard curve. The defined medium composition per liter was: 745 ml deionized water; 200 ml M-solution (in a 1 l solution contains: 42 g MOPS, 4 g tricine, 14.6 g NaCl, 8 g KOH, 2.55 g NH4Cl); 2 ml O-solution (in a 50 ml solution contains: 2.68 g MgCl2.6H2O, 1 ml T solution (in a 100

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ml solution contains: 8 ml concentrate HCl, 18.4 mg CaCl2.2H20, 64 mg H3BO4, 40 mg MnCl2.4H20, 340 mg ZnCl2, 605 mg Na2MoO4.2H2O, 10mM 57Fe solution); 2 ml Psolution (1 M KH2PO4); 1 ml S-solution (276 mM K2SO4); 0.5 ml of 0.2 % vitamin B (thiamin); 20 ml of 20 % glucose; 40 ml of 3.75 % casamino acid; and 1 ml of 100 mg/ml ampicillin. The 57Fe solution was prepared by dissolving the iron metal in 2 N H2SO4 for one week.

Cell extract of M. thermoautotrophicum used in the reduction of Isf was prepared as follows. M. thermoautotrophicum strain Marburg was grown as described (21). Five g (wet weight) cells resuspended in 50 mM Tris-Cl pH7.6 was lysed in a French pressure cell at 20,000 psi. The lysate was centrifuged for 30 min at 33,000 x g, and the resulting supernate (cell extract) was saved. All proteins were stored at ­80o C until used.

UV-visible spectroscopy and potentiometric titrations. Potentiometric measurements were performed as previously described (23, 24). All electrochemical potentials are reported relative to the standard hydrogen electrode. Isf (3 - 4 µM) was titrated at 20o C in 50 mM potassium phosphate buffer (pH 7.0 - 7.05) in a solution containing methyl viologen (0.1 mM) as the mediator dye with phenosafarin (Em = 0.252 V, pH 7.0) (5 µM) and benzyl viologen (Em= -0.362 V, pH 7.0) (5 µM) as the indicator dyes. The pH measured after the experiment was recorded as the pH for the titration. The visible spectra in each experiment were obtained and stored on an Olis-14 interfaced Cary spectrophotometer. The absorbance at 480 nm was used to monitor the amount of oxidized and reduced FMN after correcting the spectra for turbidity. The reduction potentials reported were determined by potentiometric measurements in the reductive direction. After each potentiometric titration of the FMN chromophore, the iron-sulfur flavoprotein was reoxidized completely using ferrocyanide (0.1 mM) as the mediator dye. Equilibrium of the system in the UV-visible potentiometric measurements was considered to be obtained when the measured potential drift was less than 1 mV in 5 min; this was typically around 1-2 h. The midpoint potentials (Em) and n values were calculated using the Nernst Equation indicated below, where E is the measured equilibrium potential at each point in the titration and n is the number of electrons. The

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typical error in the reported reduction potential values was ± 2-3 mV. E=Em+ (0.058/n) log ([ox]/[red]) All midpoint potential value determinations exhibited Nernstian behavior as indicated by their n values.

EPR spectroscopy and potentiometric titrations. The EPR spectra were recorded on a Bruker ESP 300E spectrometer equipped with an Oxford ITC4 temperature controller and automatic frequency counter of a Hewlett Packard Model 5340 and Bruker Gaussmeter. The spectroscopic parameters are given in the figure legends. Double integration of the EPR signals was performed with copper perchlorate (1 mM) as the standard. Isf was frozen in liquid nitrogen prior to EPR analyses.

For the power saturation studies, Isf (74 µM, pH 7.0) was reduced in the presence of 50 mM methyl viologen with a 40-fold excess of sodium dithionite prepared freshly at pH 9.0. The solution was immediately frozen in an EPR tube and stored in liquid nitrogen. Spectra of the reduced [4Fe-4S] cluster were recorded at powers varying from 0.1- 200 mW at five different temperatures between 5 and 25 K. The power for half saturation (P1/2) at each temperature was determined by a fit to a plot of log (S/P*e0.5) versus log P using Equation 7, where S is the signal amplitude, P is the microwave power incident on the cavity, and b is the inhomogeneity parameter. Best fits to the data were obtained by using a b value of 1.2. S = P /(1 + P/P1/2 )* e 0.5b The zero field splitting constant () was determined by a linear fit to a plot of ln P1/2 versus 1/T according to Equation 8, where T is the temperature, P1/2 is the power for half saturation, k is the Boltzmann constant, and A is a coefficient representative of the phonon-spin coupling properties of the [4Fe-4S] cluster. P1/2=Aexp(-/kT)

Potentiometric measurements of the [4Fe-4S] cluster were performed in an EPR-

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spectroelectrochemical cell with a quartz EPR tube describes previously (9). Isf samples (80 - 160 µM) in 50 mM potassium phosphate buffer (pH 7.0) were titrated at 20o C in the presence of the mediator dyes, 150 µM benzyl viologen (Em = -0.362 V), 150 µM methyl viologen (Em -0.440 V), 100 µM 1,1'-trimethylene-2,2'-bipyridyl (Em = -0.540 V), and 100 µM 4,4'-dimethyl-2,2'-dipyridyl (Em = -0.586 V). The intensity of the EPR signal with a g-value of 1.93 was monitored to determine the redox state of the [4Fe-4S] cluster during the titration. Potentiometric measurements were performed in the reductive and oxidative directions. The system was considered to have reached equilibrium when the measured potential drift was less than 1 mV in 2 min.

Mössbauer spectroscopy. Mössbauer spectra were recorded on a constant acceleration spectrophotometer, MS-1200D, using a Janis Super Varitemp cryostat model 8DT with a Lakeshore temperature controller model 340 and a 57Co gamma source. The experiments were done at 4.2 and 100 K. The reduced Isf was generated by adding 10fold excess of sodium dithionite.

Reduction of methanophenazine by Isf. The assay mixtures were ananerobically equilibrated with 1.0 atm of CO in a stoppered 1.0 ml-cuvette maintained at 35o C. The assay mixture (700 µl) contained: 50 mM Tris-Cl (pH 7.6), 2 mM dithiothreitol, 1 mg/l resazurine, 25 µg carbon monoxide dehydrogenase /acetyl CoA synthetase complex (CODH/ACS), and 9 µg M. thermophila ferredoxin A. After 10 min incubation, 180 µg Isf were added to the assay mixtures and incubated for 10 min. The reaction was initiated by the addition of 120 µM of 2-hydroxyphenazine (1). The absorbance at 478 nm was measured in the Hewlett Packard 8452A diode array spectrophotometer.

Reduction of Isf by M. thermoautotrophicum cell extract. The assay mixtures were anaerobically equilibrated with 1.0 atm of CO, H2, or N2 in a stoppered 1.0 mlcuvette maintained at 35o C. The assay mixture (700 µl) contained: 50 mM Tris-Cl (pH 7.6), 2 mM dithiothreitol, 1 mg/l resazurine, 180 µg M. thermoautotrophicum cell extract,

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and 13.5 µg M. thermophila ferredoxin A. After 10 min incubation, the reaction was initiated by addition of 180 µg Isf. Ferredoxin A was omitted in some assays whereas ferredoxin from Clostridium pasteurianum was substituted where indicated. The absorbance at 476 nm was measured in the Hewlett Packard 8452A diode array spectrophotometer.

RESULTS

Sequence analysis. Error in the previously reported isf sequence (14) was detected during site-directed mutagenesis studies (refer to chapter 4). Figure 1 shows the corrected sequence deduced from genomic DNA. The corrected Isf contains 191 residues, 81 fewer than previously reported. Additionally, the C-terminal residues

177 177 191

KLCDVLELIQKNRDK NSVMSWNLFRKIEIN

in the corrected Isf sequence replace in the previously reported Isf. Since the DNA library

191

containing isf gene was available, isf gene from this library was sequenced to see if there was an error that may result in error in previous study. Identical sequences were found both from genomic DNA and DNA library as reported in Figure 1. Re-sequencing revealed the correct isf sequence in pML701 used for the heterologous production of Isf reported here and previously (14).

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ATG AAA ATA ACA GGA ATT TCA GGC AGT CCA CGA AAG GGC CAG AAC M K I T G I S G S P R K G Q N TGT GAG AAA ATA ATT GGA GCT GCT CTT GAG GTT GCA AAA GAA AGA C E K I I G A A L E V A K E R

45 15 90 30

GGG TTT GAA ACT GAT ACC GTT TTT ATC TCA AAC GAG GAG GTT GCC 135 G F E T D T V F I S N E E V A 45 CCC TGC AAA GCG TGC GGG GCT TGC AGA GAT CAA GAT TTC TGT GTG 180 P C K A C G A C R D Q D F C V 60 ATT GAT GAT GAT ATG GAC GAG ATA TAT GAA AAA ATG AGG GCT GCA 225 I D D D M D E I Y E K M R A A 75 GAC GGT ATA ATT GTT GCA GCT CCC GTA TAT ATG GGG AAT TAT CCT 270 D G I I V A A P V Y M G N Y P 90 GCC CAG CTT AAA GCC CTT TTT GAC AGG AGT GTC CTG CTT CGC CGT 315 A Q L K A L F D R S V L L R R 105 AAA AAC TTT GCA CTA AAA AAT AAA GTT GGG GCA GCT CTT TCA GTT 360 K N F A L K N K V G A A L S V 120 GGG GGC TCA AGA AAC GGA GGA CAG GAA AAA ACA ATT CAG TCC ATA 405 G G S R N G G Q E K T I Q S I 135 CAT GAC TGG ATG CAC ATT CAC GGA ATG ATT GTA GTC GGC GAT AAT 450 H D W M H I H G M I V V G D N 150 TCC CAC TTC GGT GGA ATT ACG TGG AAC CCG GCA GAA GAG GAC ACT 495 S H F G G I T W N P A E E D T 165 GTT GGA ATG CAG ACA GTT TCC GAA ACT GCA AAA AAA CTC TGT GAT 540 V G M Q T V S E T A K K L C D 180 GTC CTG GAA CTT ATT CAG AAA AAT AGA GAT AAA TAA CAA AAT TCA 585 V L E L I Q K N R D K * 191 TAA CCG CAA TTT TGT CAT ATT AGA ACC GTT CCT CTC ATA AGT CTG GAT ATA CCG TAA AAA GCA ATA TTG GTT GTC TTA ATC AAA GTA TGA AGG GTT CTG TCA GGA GTA ATA TGC ATA GTT GAA TTA CAC AAA CAA TAA ATC TAT TGT AAA AAC TTA GCT CCA CCC AAA TTA ATA ATA CAG AAA TAT CCA ATC GAT TAT TGC AAA AAA AAT GAA ACA AGT TAT GGA TTT TTT CAT TAT GTA 630 675 720 765 810 825

Figure 1. Corrected nucleic acid sequence and predicted amino acid sequence of isf from M. thermophila. The DNA is presented in the 5' to 3' direction. The predicted amino acid sequence of Isf is shown in single-letter code directly below the first base of each codon. *, initial base of translation stop codon.

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EPR spectroscopy of the iron-sulfur centers. Isf was purified and sent to Dr. S.W. Ragsdale and Dr. D.F. Becker at the University of Nebraska for EPR analysis. Their results, interpretations, and conclusions follow. The reduced heterologously produced Isf displayed the rhombic EPR spectrum indicative of a [4Fe-4S]+1 type with g values at 2.06, 1.93 and an unusual split signal at 1.86 and 1.82 (Fig. 2). Additional evidence for the presence of the [4Fe-4S]1+ center is the disappearance of the g = 1.93 at the temperatures above 25 K (20). Several lines of evidence suggest the splitting signal observed at gmin region of EPR spectrum is derived from the microheterogeneity within Isf molecules. For example, the presence of viologen dyes did not contribute to this complex spectrum since the sodium dithionite-reduced sample without dyes also give a similar spectrum. Since the flavin is diamagnetic in both the oxidized and reduced state, this diamagnetic cofactor cannot give rise to this unusual feature. The possibility that a strong hyperfine interaction between an unpaired electron on the cluster and a strongly coupled proton produces a complex feature was examined. Reduced protein after an extensive exchange with D2O also exhibited the same spectrum; thus, this possibility was ruled out. In the power and temperature studies, the result showed two separate and distinct species, one with g values at 2.06, 1.92, and 1.82, and another one with g values at 2.03, 1.92, and 1.86 (refer to chapter 5). These results are consistent with the conclusion of microheterogeneity that contributes to the unusual spectrum.

Power saturation studies of the [4Fe-4S] cluster were examined at five difference temperatures (5, 10, 15, 20, and 25 K). The half saturation powers (P1/2) are in the range of 79 to 14.4 mV. The plot of P1/2 and 1/T showed a linear relationship at the temperatures higher than 5 K (Fig. 3). The zero field splitting parameter () was observed at the values between 10 and 11.5 cm-1 (Fig. 3,inset).

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Figure 2. EPR spectroscopy of the [4Fe-4S] cluster in Isf poised at various redox potentials in 50 mM potassium phosphate buffer (pH 7.0). Experimental conditions were as follows: temperature, 10 K: microwave power, 1.26 milliwatts; microwave frequency, 9.43 GHz; receiver gain, 2 x 104; modulation amplitude, 10 G; modulation frequency, 100 kHz. The derviative feature at g = 2.0 results from the mediator dyes.

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Figure 3. Semilogarithmic plot of P1/2 versus 1/T, which shows a linear relationship according to equation P1/2 = Aexp(-/kT). The slope (-/k = 1.5 +/- 0.73) yields an estimate of 11.5 cm-1 for the zero field splitting value (). Inset, a nonlinear plot of P1/2 versus 1/T, which includes the data at 5 K. The slope (-/k = 14.1 +/- 1.8) yields an estimate for of 9.8 cm-1.

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Mössbauer spectroscopy. Prior to Mössbauer spectroscopy, Isf was heterologously produced in E. coli cultured in media enriched with 57Fe. The 57Feenriched protein was purified and sent to Dr. K.K. Surerus at the University of Wisconsin Milwaukee for Mössbauer spectroscopy. Her results, interpretations, and conclusions follow. The as-purified Isf exhibited a single broad quadrupole doublet Mössbauer spectrum with an average isomer shift, = 0.45 mm/s, and an average quadrupole splitting, EQ = 1.22 mm/s, at 4.2 K (Fig. 4a). The quadrupole splitting decreased slightly at 100 K (EQ = 1.12 mm/s). Dithionite-reduced Isf exhibited a single broad nonsymmetrical quadrupole doublet with an average isomer shift, = 0.55 mm/s and an average quadrupole splitting, EQ = 1.30 mm/s at 100 K (Fig. 4b). These parameters are indicative of values for the [4Fe-4S] center with 2+ and 1+ redox states. The nonsymmetrical line shapes observed for both as-purified and reduced proteins, especially for reduced protein, suggest the irons within the cluster are not identical. Another reason may due to a microheterogeneous population of iron-sulfur clusters in the Isf molecule. The Voigt (Gaussian distribution of a Lorenzian lineshape) lineshape of the doublet, rather than Lorenzian, indicates a microheterogenous environment for the iron sites. These finding are consistent with the conclusion drawn from EPR spectrum. The property of electronic spin of the iron-sulfur cluster was studied by applying the magnetic field either parallel or perpendicular to the beam. Mössbauer spectra of the reduced Isf exhibited paramagnetic hyperfine structure indicative of reduced [4Fe4S]1+ cluster with S = ½ (Fig. 5). The shape of the spectra and the derived hyperfine structure are typical of that observed in other [4Fe-4S] proteins (4, 15, 17).

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Figure 4. Mössbauer spectra recorded at 100 K. A) oxidized Isf protein. B) reduced Isf protein. The solid line is a least-square fit with a Voigt line shape.

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Figure 5. Mössbauer spectra of reduced Isf protein recorded at 4.2 K and 450 G applied parallel (A) or 450 G applied perpendicular (B) to the beam. The solid line is a theoretical fit of an S = ½.

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Potentiometric titrations of FMN and the [4Fe-4S] center. Potentiometric measurements were performed by Dr. S.W. Ragsdale and Dr. D.F. Becker at the University of Nebraska. The absorption spectrum of Isf was followed between 300 to 700 nm during potentiometric titration (Fig. 6). Reduction of FMN was monitored at 480 nm to avoid absorption interference from the [4Fe-4S] cluster. Absorption due to the [4Fe-4S] cluster was measured at potentials below -305 mV to avoid interference from the absorption of FMN. The FMN/FMNH2 couple showed a midpoint potential of ­277 mV (Fig. 6, inset). There was no appearance of absorption around 500-600 nm, which is generally present due to the semiquinone. This destabilization of the semiquinone is a rare case for flavoprotein.

FMN has been shown to go through one electron reductions sequentially generating a semiquinone and the hydroquinone. Although the semiquinone was not detected during potentiometric titration, semiquinone may be stabilized in the physiological system. Attempts to determine the semiquinone formation in the biological system was performed in the mixture of CODH/ACS, Isf, and CO. The EPR spectra of frozen reaction mixtures at different time points were observed. The maximum of only 2.5 % of semiquinone form was observed at 28 min (Fig. 7). Over the course of 50 min, the cluster underwent reduction as the FMN was fully reduced to the hydroquinone form. The semiquinone form occurred during this reaction is classified as the anionic or red semiquinone, since the EPR signal yielded a line width with 16 gauss . Therefore, the formation of semiquinone is possible under the physiological conditions.

The Em value for the +2/+1 couple state of the [4Fe-4S] center was determined using spectroelectrochemical titration. When the data were analyzed using the Nernst equation, the Em calculated for the [4Fe-4S]2+/1+ center was ­394 mV and the slope was 53 mV (Fig. 2 and 8), results consistent with a one electron transfer carrier (the theoretical value for a one-electron transfer is 58 mV). The redox reaction was fully reversible since the reduction was titrated in both the oxidative and reductive directions. Thus, the midpoint potential of the [4Fe-4S] cluster is more than 100 mV lower than that of the FMN/FMNH2 couple (Fig. 6 and 8) and is in the range as the value reported for

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Figure 6. Potentiometric titration of the FMN in Isf (3.2 µM) in 50 mM potassium phosphate buffer (pH 7.0) at 20o C (curves 1-7, fully oxidized, -262, -272, -281, -290, 305, and ­342 mV respectively). Inset, Nernst plot of the potentiometric data.

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Figure 7. EPR spectrum of Isf (170 µM dimer) was recorded at 10 K following incubation for 17 min with CO and CODH (0.5 µM) at 25o C. The amount of FMN hydroquinone and reduced iron-sulfur cluster at this time point were 46 and 3% (0/03 spin/mol), respectively. After the sample was frozen in liquid nitrogen, the spectrum was recorded using the conditions described in figure 2)

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Figure 8. A fit of the Isf midpoint potential data to a theoretical curve generated from the Nernst equation for two redox centers with reduction potentials of ­277 mV (n = 2) and ­394 mV (n = 1).

74

other low-potential [4Fe-4S] clusters (2). These results suggest electrons flow from the 4Fe-4S center to FMN.

Potential electron acceptors for Isf. A previous study indicated ferredoxin A is a direct physiological electron donor for Isf (14); however, the physiological electron acceptor is unknown. From the properties of redox centers in Isf described above, 2electron carriers are candidates to accept electrons from Isf. Isf could accept one electron from ferredoxin A thorough the [4Fe-4S] center and two electrons are transferred to FMN. FMN in the hydroquinone form donates the 2 electrons to the 2-electron carrier. Several 2-electron carriers such as F420, NAD+, NADP, were included in reconstitution electron transport assays composed of purified components of CO, CODH, ferredoxin A, and Isf. The results showed none of these 2-electron carriers were reduced by Isf. Unless an unknown 2-electron carrier accepts electrons from Isf, these results suggest Isf is not a 1- electron 2-electron switch.

The participation of methanophenazine as electron acceptor for Isf was examined. 2-hydroxyphenazine was a gift from Dr. U. Deppenmeier. Methanophenazine was isolated from H2/CO2-grown Methanosarcina mazei Gö1 and shown to be involved in reduction of CoB-S-S-CoM (1). The as-isolated methanophenazine was water insoluble, thus 2-hydroxyphenazine (a water-soluble analogue) has been used in aqueous buffer assays (1). The midpoint potential of 2-hydroxyphenazine is ­255 mV (1). With the assumption that the redox potential of methanophenazine is similar to this, this cofactor should be able to act as an electron acceptor of Isf. However, the results revealed a different data from the theoretical assumption. Reduction of 2-hydroxyphenazine in the reconstitution electron transport system with CO, CODH/ACS, and ferredoxin A is greater than the system containing those components and Isf (Fig. 9). In the reconstitution system with CO, and CODH/ACS, no reduction of 2-hydroxyphenazine was observed. These results indicate that ferredoxin A is an electron donor for both Isf and 2-hydroxyphenazine. At this stage, we have been unable to identify the electron acceptor for Isf.

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0.5 0.4 A478 0.3 0.2 0.1 0 0 2 4 6 8 10 Minutes

Figure 9. Time course for reduction of methanophenazine with Isf from M. thermophila. The standard assay mixtures were anaerobically equilibrated with 1.0 atm. of CO in a stoppered 1.0 ml-cuvette maintained at 35 o C. The standard assay mixture (700 µl) contained: 50 mM Tris-Cl (pH 7.6), 2 mM dithiothreitol, 1 mg/l resazurine, 25 µg CODH/ACS and 120 µM methanophenazine (). The assay contained all components of the standard assay plus 9 µg M. thermophila ferredoxin A ( ). The assay contained all components of the standard assay plus 9 µg M. thermophila ferredoxin A and 180 µg Isf ( ).

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Evidence for Isf homologs in phylogenetically and physiologically diverse microbes. Two genomic sequences of the CO2-reducing methanoarchaea, M. jannaschii and M. thermoautotrophicum, were recently completed (3, 22). M. jannaschii and M. thermoautotrophicum are phylogenetically and physiologically distinct from M. thermophila. Neither M. jannaschii or M. thermoautotrophicum can utilize acetate as growth substrate, instead they evolve methane by reducing CO2 using H2 as electron donor. Two open reading frames (ORF) from M. jannaschii, MJ0731 and MJ1083, which encode 192 and 194 amino acid proteins have 40 and 49 % identity to Isf from M. thermophila. The genome of M. thermoautotrophicum contains three ORFs, MTH135, MTH1473, and MTH1595 with 41, 34, and 30 % identity to the M. thermophila Isf. Comparisons of these Isf sequences also show a completely conserved N-terminal cysteine motif (Fig. 10). These results suggest a general function for this electron carrier in the methanoarchaea; thus, Isf-like proteins may be widespread electron carriers for methanogenesis in diverse methanoarchaea. An electron carrier function of Isf in methanoarchaea other than M. thermophila was examined to test this notion. Extracts of H2/CO2 grown M. thermoautotrophicum were able to catalyze the reduction of M. thermophila Isf with either H2 or CO as the electron donor (Fig. 11). These results suggest Isf homologues are components of the electron transport chain in CO2reducing methanoarchaea. The reduction of Isf in the presence of CO is higher than that of H2 suggesting Isf may be specific for electron transport coupled to CO oxidation. The Isf reduction rate was stimulated by addition of ferredoxin A from M. thermophila. This implies that ferredoxin A or a homologue is able to couple the oxidation of either H2 or CO to the reduction of Isf. However, ferredoxin from C. pasteurianum cannot replace ferredoxin A for Isf reduction in this system (Fig. 12); thus, the reaction appears to be ferredoxin A specific.

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Figure 10. Multiple amino acid sequence alignment of Isf from M. thermophila with sequences deduced from open reading frames identified in the genomic sequences of M. jannashii and M. thermoautotrophicum. Accession numbers for the M. jannashii and M. thermoautotrophicum sequences and percentage identity (in parenthesis) with M. thermophila Isf are as follows: MTH135 (41%), MTH1473 (34%0, MTH1595 (30%), MJ 1083 (49%), and MJ0731 (40%). Asterisks indicate conserved cysteine residues.

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Figure 11. Time course for reduction of Isf with extract from M. thermoautotrophicum. The assay mixture (700 µl) contained cell extract (180 µg protein), M. thermophila ferredoxin (13.5 µg), 50 mM Tris (pH 7.6) and 2 mM dithiothreitol. Ferredoxin was omitted in two of the assays ( , ). The assay mixtures were anaerobically equilibrated with 1 atm. of CO ( , ·), H2 ( ,,), or N2 () in a stoppered 1.0-ml cuvette maintained at 35o C. After a 10-min incubation, the reaction was initiated be the addition of 180 µg of Isf.

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0.20

0.15

A476

0.10

0.05

0.00 0 5 10 Minutes 15

Figure 12. Time course for reduction of Isf with extract from M. thermoautotrophicum. The assay mixture (700 µl) contained: cell extract (180 µg protein), either M. thermophila ferredoxin A (·, ) or C. pasteurianum ferredoxin (o, ) (13.5 µg), 50 mM , ) in a stoppered 1.0-ml cuvette

Tris (pH 7.6) and 2 mM dithiothreitol. The assay mixtures were anaerobically equilibrated with 1 atm. of CO (·, o), or H2 ( of 180 µg of Isf. maintained at 35o C. After a 10-min incubation, the reaction was initiated by the addition

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DISCUSSION

The properties and physiological role of Isf was examined in this investigation. Previous studies indicate an involvement of Isf in the electron transport chain of COdependent CoM-S-S-CoB during methanogenesis of M. thermophila (14). However, further characterization of the redox centers in Isf is necessary to provide insight into a specific role for Isf in the electron transport chain. The iron-sulfur cluster type present in the heterologously produced Isf was unequivocally demonstrated by EPR and Mössbauer spectroscopy to be [4Fe-4S]. The Isf sequence reveals a completely conserved cysteine motif which has the potential to serve as ligands for the [4Fe-4S] center; however, the cysteine spacing in the motif is distinct from any known motifs accommodating known [4Fe-4S] centers. The unusual cysteine motif in Isf may represent a novel class of cysteine motif ligating the [4Fe-4S] center among the Isf-like sequences. The presence of two negative absorption features in the EPR spectra of the reduced cluster is unusual. The results presented here indicate that microheterogeneity within the population of Isf molecules accounts for this atypical feature. This situation is similar to that of a class of corinoid/iron-sulfur proteins from methanoarchaea and homoacetogenic anaerobes from the Bacteria domain in which the reduced [4Fe-4S] cluster exhibits a broad absorption feature in the same g value region (10, 12, 19). The Em results for the 4Fe-4S center and FMN indicate that the intra-electron transfer from [4Fe-4S] to FMN is plausible. The Em values from this investigation and other known Em values from other electron transfer components are consistent with the electron flow as CODH (Em for center C and [4Fe4S] center = -540 and ­444 mV, (16)) Õ ferredoxin (Em = -407 mV, (5)) Õ Isf [4Fe-4S] (Em = -394 mV) Õ Isf [FMN] (Em = -277 mV) Õ an unknown electron carrier (Fig. 13). Most flavin/Fe-S proteins stabilize four redox states: flavinox:FeSox, flavin semiquinone:FeSox, flavin semiquinone:FeSred, and flavinred:FeSred (13, 18). Stabilization of the semiquinone allows versatility of mediating both one-electron and two-electron transfer reactions. The results reported here suggest only a transient stabilization of the FMN semiquinone; however, stability of semiquinone may be increased when Isf interacts with its physiological electron donors/acceptors.

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CdhA FdxA "C" -540 mV "B" -444 mV CO -518 mV CO2

4Fe4S -407mV Isf 4Fe4S -395 mV FMN -277 mV

?

Figure 13. Proposed electron transport pathway for oxidation of CO or the carbonyl group of acetyl-CoA. Cdh A, subunit of the CODH/ACS; FdxA, ferredoxin A; ?, postulated unknown electron carrier. Midpoint potentials are shown in mV.

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Due to the high instability of the semiquinone of FMN, it is possible that the physiological electron acceptor for Isf (which is unknown) could be a two-electron carrier and Isf functions as a one-electron/two-electron switch. However, involvement of the obligate two-electron carrier coenzyme F420, NAD, NADP in the electron transport chain has been excluded. A role for the two-electron carrier methanophenazine (1) as electron acceptor of Isf was ruled out. Indeed, methanophenazine competed with Isf for ferredoxin A. Methanophenazine has never been isolated from acetate-grown M. thermophila cells. Due to the reduction of methanophenazine by ferredoxin A of M. thermophila, it is possible that this compound is present in cells and may function in place of Isf under different growth conditions. Additional experiments are required to confirm if the reduction of methanophenazine is physiologically significant.

The environment in the FMN binding site of Isf must be different from other flavoproteins. To stabilize a negative charge of hydroquinone, an amino acid with positive charge may be required. From the sequence comparisons, two positively charged amino acids, K94 and R124, are highly conserved. These two residues may flank the FMN binding site and would result in the thermodynamic stabilization of the hydroquinone.

The presence of Isf-like sequences in the genomes of M. jannaschii and M. thermoautotrophicum, and the use of either H2 or CO as electron donor for Isf reduction by M. thermoautotrophicum cell extract, imply a physiological significance of Isf as a primary electron carrier in different methanogenesis pathways. The greater reduction of Isf using CO as electron donor and the ability of M. thermoautotrophicum to grow and produce CH4 with CO as the sole energy source (6) indicate a physiological role for CODH in the energy metabolism. Furthermore, this methanoarchaeon involves a CODH in the synthesis of CO for incorporation into acetyl-CoA for cell carbon (11). The CODHs from either M. thermoautotrophicum or M. jannaschii have not been purified and, therefore, the electron acceptor is unknown. The results presented here are consistent with ferredoxin as the electron acceptor; however, purification of the CODH is necessary to prove this hypothesis. The implicated functions of Isf homologues in

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diverse methanoarchaea are also supported by the gene organization in M. jannaschii. The MJ0731 is located adjacent to MJ0722 and MJ0728, which encode an 8Fe ferredoxin- and CODH/ACS-like proteins (3). However, there is no such gene arrangement found in M. thermoautotrophicum (22).

ACKNOWLEDEGEMENT

I would like to thank Dr. D.F. Becker and Dr. S.W. Ragsdale at University of Nebraska-Lincoln for performing the EPR spectroscopy, and Dr. K.K. Surerus at University of Wisconsin Milwaukee for Mössbauer spectroscopy. I also thank Dr. U. Deppenmeier for the 2-hydroxymethanophanize and Dr. J. M. Bollinger for the suggestion on 57Fe solution preparation. Work described here was partially supported National Institutes of Health Grant No. 1-R15-GM52666-01 (KKS), and by Department of Energy Basic Energy Sciences Grants No. DE-FG02-ER20053 (SWR) and DE-FG0295ER20198 (JGF). UL was supported by a MOSTE grant from Thailand.

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Bruschi, M., and F. Guerlesquin. 1988. Structure, function and evolution of bacterial ferredoxins. FEMS Microbiol. Rev. 4:155-75.

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Christner, J. A., P. A. Janick, L. M. Siegel, and E. Munck. 1983. Mössbauer studies of Escherichia coli sulfite reductase complexes with carbon monoxide and cyanide. Exchange coupling and intrinsic properties of the [4Fe-4S] cluster. J. Biol. Chem. 258:11157-64.

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Clements, A. P., L. Kilpatrick, W.-P. Lu, S. W. Ragsdale, and J. G. Ferry. 1994. Characterization of the iron-sulfur clusters in ferredoxin from acetategrown Methanosarcina thermophila. J. Bacteriol. 176:2689-2693.

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Daniels, L., G. Fuchs, R. K. Thauer, and J. G. Zeikus. 1977. Carbon monoxide oxidation by methanogenic bacteria. J. Bacteriol. 132:118-126.

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Ferry, J. G. 1992. Biochemistry of methanogenesis. Crit. Rev. Biochem. Mol. Biol. 27:473-503.

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Ferry, J. G. 1992. Methane from acetate. J. Bacteriol. 174:5489-5495. Harder, S. R., B. A. Feinberg, and S. W. Ragsdale. 1989. A

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spectroelectrochemical cell designed for low temperature electron paramagnetic resonance titration of oxygen-sensitive proteins. Anal. Biochem. 181:283-7. 10. Harder, S. R. L., W. P. Lu, B. A. Feinberg, and S.W. Ragsdale. 1989. Spectroelectrochemical studies of the corrinoid iron-sulfur protein involved in acetyl coenzyme-A synthesis by Clostridium thermoaceticum. Biochemistry. 28:9080-9087. 11. Hemming, A., and K. H. Blotevogel. 1985. A new pathway for CO2 fixation in methanogenic bacteria. Trends Biochem. Sci. 10 :198-200. 12. Jablonski, P. E., W. P. Lu, S. W. Ragsdale, and J. G. Ferry. 1993. Characterization of the metal centers of the corrinoid;iron- sulfur component of the CO dehydrogenase enzyme complex from Methanosarcina thermophila by EPR spectroscopy and spectroelectrochemistry. J. Biol. Chem. 268:325-329. 13. Johnson, M. K. 1994. Iron-sulfur proteins. Encyclopedia of Inorganic Chemistry, Johnson Wiley and Sons (King, R.B., ed). 4:1896-1915. 14. Latimer, M. T., M. H. Painter, and J. G. Ferry. 1996. Characterization of an iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol. Chem. 271:24023-24028. 15. Lindahl, P. A., E. P. Day, T. A. Kent, W. H. Orme-Johnson, and E. Munck. 1985. Mössbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2. J. Biol. Chem. 260:11160-73. 16. Lu, W. P., P. E. Jablonski, M. Rasche, J. G. Ferry, and S. W. Ragsdale. 1994. Characterization of the metal centers of the Ni-Fe-S component of the carbonmonoxide dehydrogenase enzyme complex from Methanosarcina thermophila. J. Biol. Chem. 269:9736-9742. 17. Middleton, P., D. P. Dickson, C. E. Johnson, and J. D. Rush. 1978. Interpretation of the Mössbauer spectra of the four-iron ferredoxin from Bacillus stearothermophilus. Eur. J. Biochem. 88:135-41. 18. Muh, U., I. Cinkaya, S. P. Albracht, and W. Buckel. 1996. 4-HydroxybutyrylCoA dehydratase from Clostridium aminobutyricum: characterization of FAD and iron-sulfur clusters involved in an overall non-redox reaction. Biochemistry.

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35:11710-8. 19. Ragsdale, S. W., P. A. Lindahl, and E. Munck. 1987. Mössbauer, EPR, and optical studies of the corrinoid;iron-sulfur protein involved in the synthesis of acetyl-CoA by Clostridium thermoaceticum. J. Biol. Chem. 262:14289-14297. 20. Rupp, H., K. K. Rao, D. O. Hall, and R. Cammack. 1978. Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation. Biochim. Biophys. Acta. 537(2):255-60. 21. Schonheit, P., J. Moll, and R. K. Thauer. 1980. Growth parameters (Ks, mumax, Ys) of Methanobacterium thermoautotrophicum. Arch. Microbiol. 127:5965. 22. Smith D. R. , L. A. Doucette-Stamm, C. Deloughery, H. Lee, J. Dubois, T. Aldredge, R. Bashirzadeh, D. Blakely, R. Cook, K. Gilbert, D. Harrison, L. Hoang, P. Keagle, W. Lumm, B. Pothier, D. Qiu, R. Spadafora, R. Vicaire, Y. Wang, J. Wierzbowski, R. Gibson, N. Jiwani, A. Caruso, D. Bush, H. Safer, D. Patwell, S. Prabhakar, S. McDougall, G. Shimer, A. Goyal, C. J. Daniels, J. I. Mao, P. Rice, J. Nölling, and J. N. Reeve. 1997. The complete genome sequence of Methanobacterium thermoautotrophicum strain H: functional analysis and comparative genomics. J. Bacteriol. 179:7135-7155. 23. Stankovich, M., and B. Fox. 1983. Redox potentials of the flavoprotein lactate oxidase. Biochemistry. 22:4466-72. 24. Stankovich, M. T. 1980. An anaerobic spectroelectrochemical cell for studying the spectral and redox properties of flavoproteins. Anal. Biochem. 109:295-308. 25. Terlesky, K. C., and J. G. Ferry. 1988. Purification and characterization of a ferredoxin from acetate-grown Methanosarcina thermophila. J. Biol. Chem. 263:4080-4082. 26. White, R. H., and D. Zhou. 1993. Biosynthesis of the coenzymes in methanogens. Methanogenesis, J.G. Ferry, ed. Chapman and Hall, New York. 409-444.

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CHAPTER 5 A NOVEL 4Fe-4S CLUSTER BINDING MOTIF IN THE IRONSULFUR FLAVOPROTEIN OF Methanosarcina thermophila

ABSTRACT

Isf (Iron-sulfur flavoprotein) from Methanosarcina thermophila has been produced in Escherichia coli as a dimer containing two [4Fe-4S] clusters and two FMN (flavin mononucleotide). The deduced sequence of Isf contains six cysteines (C16, C47, C50, C53, C59, and C180), four of which (C47, C50, C53, and C59) compromise a motif perfectly conserved among several putative Isf sequences available in the databases. The spacing of the four conserved cysteines is highly compact and atypical of motifs coordinating known 4Fe-4S clusters; therefore, all 6 cysteines in Isf were altered to either alanine or serine to obtain biochemical confirmation that the motif coordinates the 4Fe4S cluster and to determine the influence of the protein environment on the properties of the cluster. All except the C16S variant were produced in inclusion bodies that required solubilization and reconstitution of the iron-sulfur cluster and FMN. The UV-visible spectra of all variants indicated the presence of iron-sulfur clusters and FMN. The reduced C16X (X = A or S) variants showed the same EPR spectra as wild type Isf whereas the reduced C180X variants showed EPR spectra similar to one of the 4Fe-4S species present in the wild type Isf spectrum. EPR spectra of the oxidized C50A and C59A variants showed g-values characteristic of a 3Fe-4S cluster. The spectra of the C47A and C53A variants indicated a 4Fe-4S cluster but with g-values different from wild type. The retention of 4Fe-4S cluster in the C47A and C53A variants suggests functional replacement of the cysteines by 2-mercaptoethanol that was present in the reconstitution buffer. The reduced C47S, C50S, and C53S exhibited EPR spectra of 4Fe-4S centers. EPR spectrum of both C47S and C53S revealed two different 4Fe-4S ligand species, which could be due to the replacement of the missing ligand by serine or 2mercaptoethanol. Taken together with strict sequence conservation, these results indicate that C47, C50, C53 and C59 are ligands to the 4Fe-4S cluster, a result which identifies the most compact cysteine motif know which ligates a redox-active 4Fe-4S cluster. The

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results suggest C16 is important for maintaining a local conformation required for transfer of electrons from ferredoxin A to FMN, and that C180 is essential for the overall structural integrity of Isf. The reduction of FMN in the Isf variants by ferredoxin A was either several-fold impaired or enhanced suggesting that the 4Fe-4S cluster serves to transfer electrons from ferredoxin A to FMN.

INTRODUCTION

Two-thirds of the biologically produced methane in nature originates from the methyl group of acetate in a pathway where acetate is cleaved and the methyl group is reduced to methane with electrons derived from oxidation of the carbonyl group to carbon dioxide (7). Much is known concerning the cleavage of acetate and one-carbon transfer reaction (6); however, less is known regarding electron transport. Recently, a novel iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was characterized which participates in electron transport during the methanogenic fermentation of acetate (2, 13). The homodimeric Isf contains two FMN molecules and two 4Fe-4S clusters unequivocally identified by EPR and Mössbauer spectroscopy. The midpoint-potential values of the 4Fe-4S cluster and FMN are -394 and -277 mV, respectively. These results are the basis for a postulated role for the cluster in electron flow from ferredoxin A, the physiological electron donor for Isf, to the 4Fe-4S cluster and then to the FMN of Isf, however, this proposal has not been tested. The physiological electron acceptor for Isf is unknown. The deduced sequence of Isf contains six cysteines, four of which are in a highly compact novel motif (CX2CX2CX4-7C) that is perfectly conserved among putative Isf-like sequences identified in the databases suggesting the motif ligates the 4Fe-4S cluster.

The cubane [4Fe-4S] cluster is ubiquitous in proteins from all domains of life (22) where they mainly function in electron transfer. The sulfur atom of cysteine is the prominent protein ligand coordinated to iron atoms in these clusters. Few examples of variations from cysteine ligation include aconitase with oxygen ligation originating from hydroxide, water, or substrate. The 4Fe-4S cluster in the ferredoxin from Pyrococcus

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furiosus is ligated with oxygen from aspartate (27). An iron atom in the 4Fe-4S cluster of hydrogenase from Desulfovibrio gigas is coordinated by a histidyl nitrogen (26). A single motif (CX2CX2C plus a distal C in the polypeptide chain) coordinates all low potential, redox active, 4Fe-4S clusters for which cysteine is the exclusive ligand. Possible exceptions to this ubiquitous 4Fe-4S motif are found in the corrinoid/iron sulfur proteins of M. thermophila and Clostridium thermoaceticum, and a putative iron-sulfur protein from Rhodobacter capsulatus, where the sequence CX2CX4CX16C is perfectly conserved (14); however, conclusive evidence for involvement of this motif in ligation of 4Fe-4S clusters has not been reported. Thus, the highly conserved CX2CX2CX4-7C motif in Isf is the most compact motif known with the potential to coordinate a 4Fe-4S motif. Although the great majority of iron-sulfur proteins function in electron transfer reactions, the clusters in a few function in non-redox catalysis or serve a structural role. Still other ironsulfur clusters bind nucleic acids or play a regulatory role (3, 10). Two of these, endonuclease III and MutY, contain a redox inert 4Fe-4S cluster coordinated by a compact cysteine motif (CX6CX2CX5C) (19, 20). Although the 4Fe-4S cluster of Isf has reversible redox activity, conclusive evidence for a role in electron transfer has not been reported.

Much has been learned regarding the coordination of iron-sulfur clusters utilizing site-specific replacement of residues ligating the clusters. Changes in spectroscopic properties and other characteristics have provided information regarding the polypeptide environment of the cluster and the effects that the coordinating ligands have on the biochemical properties of the cluster. Thus, a series of site-specific replacements in Isf were performed to obtain biochemical evidence for the novel putative cysteine motif and further characterize the biochemical and physiological properties of the 4Fe-4S cluster. The results support involvement of the remarkably compact motif in coordination of the 4Fe-4S cluster that is surprisingly resistant to changes in ligation. The results also support a role for the 4Fe-4S cluster in the transfer of electrons from the physiological electron donor (ferredoxin A) to FMN.

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Portion of the results contained in this section were obtained by Dr. J.H. Golbeck and M. L. Antokine at Pennsylvania State University. Their contributions are noted in the appropriate sections.

EXPERIMENTAL PROCEDURES

Sequence comparisons. Microbial genomic sequence databases were searched at http://www.tigr.org. Sequences were aligned using the program Clustal X version 1.64b.

Plasmid construction and site directed mutagenesis. Plasmid pML701, which contains the entire gene for Isf, was used as a template to construct mutants. Site directed mutagenesis was performed using MORPH as described by the manufacturer (5 Prime 3 Prime, Inc.® 5603 Arapahoe, Boulder, CO 80303). Each construct was confirmed for the intended mutation by sequencing using the automated dideoxy method at the Penn State University nucleic acid facility.

Protein production and purification. Escherichia coli BL21 (DE3) cells transformed with derivative expression plasmids carrying the designated isf mutations were grown on LB broth supplemented with 100 µg/ml ampicillin. Once cells reached an A600 of about 0.8, they were induced to produce high levels of the Isf variants by addition of 1% (final concentration, w/v) Bacto-lactose for 2 h. The cells were harvested by centrifugation at 11,800 x g for 10 min at 4oC. The cell pellets were frozen at -70oC. The C16S variant and wild type was purified as described (13). All other variants were purified as follows. Approximately 5 g (wet weight) of cells were suspended in 6 volumes (w/v) of buffer A (50 mM Tris-HCl pH 7.6, 200 µg/ml lysozyme, and 2 mM DTT) and incubated for 20 min at 21oC. Cells were lysed by two passages through a French pressure cell at 20,000 psi. The lysate was centrifuged at 10,000 x g for 30 min at 4oC. The pellet, containing inclusion bodies, was washed twice in 30 ml buffer B (50 mM Tris-HCl, pH 7.6, 2 M urea, 1% Triton X-100, 2 mM DTT). The protein aggregates were solubilized in 2 ml buffer C (50 mM Tris-HCl, pH 7.6, 6 M guanidine-HCl), and incubated for 2 h at 21oC. Insoluble protein was removed by centrifugation at 10,000 x g

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for 10 min at 4oC. The protein solution at this stage is termed "denatured". The soluble fraction was then diluted 100-fold in buffer D (50 mM Tris-HCl, pH 7.6, 500 mM Larginine, 2 mM DTT), and incubated at 4oC for 12 h. In the following step, the sample was concentrated using PEG 8000. The protein was dialyzed in buffer E (50 mM TrisHCl, pH 7.6, 250 mM L-arginine, 200 mM NaCl, 2 mM DTT) and then F (50 mM TrisHCl, pH 7.6, 200 mM NaCl, 2 mM DTT). The protein at this stage is defined as "renatured". There was no apparent change in subunit size among wild type and the variants as judged by migration in SDS-PAGE. The overall procedure resulted in homogenous proteins as judged by SDS-PAGE.

Ethylenediaminetetraacetic acid (sodium salt) (EDTA)-treated wild type protein was prepared by incubated 6 mg of as-purified wild type in buffer G (50 mM Tris-Cl pH7.6, 400 mM NaCl, 2 mM EDTA and 2 mM DTT) for at 21o C. Then the EDTAtreated protein was treated in buffer D, E, and F as stated above.

Reconstitution of iron-sulfur clusters and FMN into renatured apoprotein. Reconstitution of iron-sulfur clusters and FMN was performed by adding 1 ml of 10 mM FMN, 800 µl of 2-mercaptoethanol, 300 µl of 60 mM FeCl3, and 300 µl of 60 mM Na2S to 100 ml renatured apoprotein solution (12 mg). All reagents were added drop-wise with 10 min intervals between steps, and the reconstitution reaction was incubated at 4oC for 12 h. The protein was concentrated with an ultrafiltration unit fitted with a YM 30 membrane (Amicon, Beverly, Mass.) and the unbound molecules were removed by a PD10 gel filtration. The protein at this step is called "reconstituted". The recovery yield after the overall processes (denatured, renatured, and reconstituted processes) was varied, and in the range of 20-50 %.

Spectroscopy. UV-visible spectra were obtained with a Hewlett-Packard 8452A diode array spectrophotometer. EPR signals of iron sulfur clusters were recorded with a Bruker ECS 106 Electron Paramagnetic Resonance (EPR) X-band spectrometer operating with an ER/4012 ST resonator and an Oxford liquid helium cryostat. The temperature

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was controlled using an ITY4 Oxford temperature controller. The microwave frequency was determined with a Hewlett-Packard 5340A frequency counter.

Reduction of Isf by ferredoxin A. Experiments were carried out in a stoppered 1.0 ml-cuvette equilibrated with an atmosphere of CO. Continuous reduction of ferredoxin A was accomplished by including catalytic amounts of CODH/acetylCoA synthetase. All protein components except the Isf variants were anaerobically purified as previously described (13, 23, and 24). The assay mixture contained 27 µg CODH/acetylCoA synthetase and 9 µg ferredoxin A in anaerobic 50 mM Tris-HCl (pH 7.6) containing 500 mM sucrose, 0.1 mg/l resazurine, and 2 mM DTT. After 10 min incubation, 180 µg of the indicated Isf variant was added to the assay mixtures to initiate the reaction. The absorbance at 476 nm was measured to follow the reduction of FMN without interference from reduction of the iron-sulfur cluster. Ferredoxin A and CODH/acetyl CoA synthetase were present in catalytic amounts such that any absorption change in these proteins did not interfere with the assay.

RESULTS Sequence comparisons of Isf from M. thermophila with putative Isf proteins. Figure 1 shows that metabolically diverse species contain sequences with identity to M. thermophila Isf suggesting the possibility that this electron carrier functions in carbon dioxide-reducing (Methanococcus jannaschii and Methanobacterium thermoautotrophicum) and sulfate-reducing (Archaeoglobus fulgidus) Archaea, and also in metabolically diverse procaryotes from the Bacteria domain (Chlorobium vibrioforme, Clorobium tepidum, and Clostridium difficile). Only Isf from M. thermophila has been characterized; thus, the sequences shown in Figure 1 are putative Isf homologs. Nonetheless, comparison of these sequences with Isf from M. thermophila shows an unusually compact N-terminal cysteine motif with a strictly conserved spacing of CX2CX2CX4-7C atypical of cysteine motifs required for 4Fe-4S coordination. The strict conservation provides a strong indication for involvement in ligation of the 4Fe-4S cluster; however, it is possible that any one of these conserved cysteines is essential for

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16

47

MST MCJ-2 MBT-1 MBT-2 AF-2 MCJ-1 CV CT AF-3 MBT-3 AF-1 CD MST MCJ-2 MBT-1 MBT-2 AF-2 MCJ-1 CV CT AF-3 MBT-3 AF-1 CD

---------M ---------M MKQKEVDFMV ---------------------------M ---------M ---------M ---------M ------------------M ---------M

50 53

KITGISGSPR KVIGISGSPR KVIGICGSPR MILGICGSPR MIVGISGSPR KVFGISGSPR KVIGINGSPR KVIGINGSPR KLLAINGSPN ---------KAVGILGSPR IITVMNGSPR

59

KGQNCEKIIG PEGNTTLLVR KNGNTEILLR K-QATEHVLE R-KATEFVLG L-QGTHFAVN PAGNTSIMLK RAGNTSIMLK K-RNTLFLLE ------MVLE KYGNASKMLD KNGATSKVLT EIYEKMRAAD EILKKMKEAD RVVELAASAH PLYELLRRAA ELYEMLKDAK EVYENLIWAD EIFEKMVEAN EIFAKMVEAD KVLEKMQEAD DIIDRIRDSE ELKRLVEESD YIHDIITKSD

AALEVAKERG EALNAIAEEG EALDAAEEAG RALSMLEDDG EALKMLEERG YALNYLKEKG TVFETLEQEG TIFEVLEDEG VIAEEVKKLG HCRDAIESHG AALKELENSG YLYKDIERLI GIIVAAPVYM GIILGSPVYF GIIIGSPVYF GIIIATPVYN GIVMATPVYN GVIIGTPVYQ GMILGSPVYF GMILGSPTYF AIVIGTPTYF GFIVATPVYF AVILASPVYY GVIFGSPTYG

FETDTVFISN IETEFISLAD AETELVRLAG LETEFFTVRG FETKFFTVRG AEVRYFSVSR IETELIQVGG IETELIQVGG HEAEIIHLKD VETDIISLRG FEVEKVHISS PDVKINYFDL GNYPAQLKAL GGVSAQLKML GSVTAQTKMF GGVSAQIKAI GGVSAQIKAV GNVTGQLKTL ADITPELKAL ADITPELKAL GNVTGIVKNL GTARGDLMAA LNVTAQMKTF SSVTGLFKVF

EEVAP--CKA KELNP--CIG LDINP--CRA KNISP--CRH KKISP--CQH KKINF--CLH TDIKG--CRA TNIKG--CRA YEIKE--CKG MKIES--CRA KKINY--CTG SEVNPSYCIG FDRSVLLRRMDRSRPLR-MDRTRPLR-MDRCRALGAE MDRCRALVAA MDRCRAILAK IDRSGFVSRT IDRAGFVSRT IDRSRMAR-M LQRIGMVSRA IDRMLPYG-TDRAHMML--

49 49 58 47 47 48 49 49 48 32 49 51 105 104 113 104 104 106 108 109 103 89 104 107

CGACRDQDFCNMCKEEGKCDSCKKTGECDYCLRNKECDYCLKHKECDYCIKKKEG CYACIRNKNS CYACIKNKNS CDACLKGD-CLSCAKKHRCGTCLAKGECLNCYKMGK-

-CVID-DDMD -CPII-DDVD -CAIE-DDLN -CVLK-DDMF -CRIK-DDMF -CIHK-DDME KCSTK-DGFN ECSTKGDGFN -CSQK-DDIY -CRID-DGLN -CVQR-DDMD -CINQNDKVE

MST MCJ-2 MBT-1 MBT-2 AF-2 MCJ-2 CV CT AF-3 MBT-3 AF-1 CD MST MCJ-2 MBT-1 MBT-2 AF-2 MCJ-1 CV CT AF-3 MBT-3 AF-1 CD

KNFALKNKVG IGFQLRNKVG SEFRLANRVG DYDSLRGKVG DYDFFRGKVG NPKVLRGRVG NGQLFRHKVG NGQLFRHKVG GNYRLRNRVF SDGFLSWKVG HRPTLKGKYG ERLLYRKPCI NPAE-----GKAP-----GGAK-----SRDT-L---SRDT-L---SKDRGK---GRDG-----GRDG-----QGDAGW----WAP-----KKASQLGSKI NQNP------

AALSVGGSRN GAVAVGASRN GAVTVGGSRN MGIAVGGDRC MAIAVGGDRI MAIAVGGDRN ASIVS--LRR ASVVS--LRR APVVTSGLRN GPIAV--ARR GSIVVY-AGV AVTTY--ENA EDTVGMQTVS GDCKNDDIGL GESADDMTGI EVLKRTHMDS EGVKEDEEGF KGVEEDEEGL GEVVNDTEGM GEVVNDTEGM RSVKKDEIAI GEVEDDSEGI AEAFESKYRM ----------

GGQEKTIQSI GGQETTIQQI GGQETACRDI GGQEPALMQI GGQELAIQQI GGQEIALRTI GGGVHAYDSI GGGIHAYDSI GGAEYAAMSL GGHTATIQEL GKPEEVAGYM RGS-KAISFI E-TAK--KLC E-TAR--NLG E-TAR--NLG KPSKRPWACL R-SLR--KTV R-VLR--KTL D-NMR--DLG E-NMR--DLG N-SAK--ALA E-TIR--RFG EPSDEDLELQ ----------

HDWMHIHGMI HNFFLIHSMI HSFFLIHEAA HTFYILNGVI LTFYILNGVI HDFFIINEMI NHLFQICQMF NHLFQICQMF IVYALGQAML LMFYFINDMI NRVLKAWGIV KSMVLDSGGY D-----VLEL K-----KVAE R-----RVAL KGSWTLKDPE K-----RFAE N-----RFYE K-----SMAF H-----SMAF KR--IVEVAE E-----NVAE K-----QLLT ----------

VVGDNS---VVGDND-PTA VVGNAS-PTA PVSGGS-FGA PVSGGS-FGA PVGGGS-FGA MVGSTY---W MVGSTY---W PVSIVE-NPI VPGSTY-WNM PVGYAVGFGV VCGSLS---I IQKNR----VVKLI----LAARI----ILFYS----MLEKM----VLKEK----LLKKL----LLK------ATKNL----LIKRI----LIKNYGHLMK ----------

HFGGI---TW HYGGT---GV HYGGT---GV NLGAC---FW NIGAT---FW NLGAT---FW NLG-----FG NLG-----FG TTGTFPVGVI VFG------IPGEVGDEDL KTG------F -------DK-------KK-------HG-------EFI -------EGV -------RGL -------NAS ----------------RES -------NGG ADYEFWKEKG ----------

158 160 169 160 160 162 158 159 162 139 163 155 ------SFI -191 193 202 203 195 198 192 188 201 173 220 159

180

Figure 1. Multiple amino acid sequence alignment of Isf from M.thermophila (MST) with sequences deduced from open reading frames identified in the genomic sequences of M. jannaschii (MCJ), M. thermoautotrophicum (MBT), Archaeoglobus fulgidus (AF),

94

Chlorobium vibrioforme (CV), Chlorobium tepidum (CT), and Clostridium difficile (CD). The numbers after the abbreviated name of organisms indicate the different protein isoforms. Database codes for each protein are as follows, MST: Genbank U50189; MCJ-1, -2 : Genbank C64391 (MJ0731), B64435 (MJ1083); MBT-1,-2,-3: Genbank AE000802 (MTH135), AE000908 (MTH1473), AE000919 (MTH1595); AF1-,-2,-3: AE0010041 (AF1438), AE0009971 (AF1519), AE0009721 (AF1896); CV: EMBL Z83933.1; CT: C tepidum gct10; CD: CD shotgun.dbs cd2h6.q1t. Cysteines (C16, 47, 50, 53, 59, and 180) in Isf of M.thermophila are numbered at the top line. Residues conserved in at least 7 out of 10 sequences are shaded in gray. Putative FMN binding regions in Isf are underlined.

95

another function and other non-cysteinyl residues may ligate the 4Fe-4S cluster. Thus, we undertook a biochemical approach to obtain experimental evidence for the proposed role of the motif and investigate properties of the cluster dependent on the protein environment.

Heterologous production, purification and reconstitution of wild type Isf and variants. In the course of the investigation of cysteine ligation in the 4Fe-4S cluster, six cysteines present in Isf (Fig. 1) were individually altered to either alanine or serine. Except for C16S, the variants were contained in inclusion bodies. The soluble C16S was purified the same as for wild type. The inclusion bodies were isolated by centrifugation as a first step in purification of the remaining variants. The isolated inclusion bodies were extracted in guanidine-hydrochloride to solubilize the proteins. At this juncture, no discrete bands were detected by native PAGE (data not shown) suggesting the proteins were denatured. The solubilized variants were diluted in buffer containing arginine to prevent protein aggregation during renaturation (1, 5, and 25). After removal of the arginine by dialysis, native PAGE indicated no discrete bands (data not shown) suggesting the proteins had not achieved the native state. UV-visible spectra indicated the proteins contained very low amounts of iron-sulfur clusters and FMN (data not shown); thus, the apoproteins were incubated in the presence of ferric iron, sulfide, and FMN to reconstitute the redox clusters. UV-visible spectroscopy (Fig. 4, 5) indicated incorporation of flavin and iron-sulfur clusters. Native PAGE (Fig. 2) indicated a discrete band for each variant migrating to approximately the same position as the purified wild type, which suggested all are dimeric in accord with the initial characterization of the wild type (13). These results suggested that either an iron-sulfur cluster or flavin, or both, must be present to adopt a native conformation. There was no apparent change in subunit size among wild type and the Isf variants as judged by migration in SDS-PAGE (data not shown). A similar denaturation/renaturation/reconstitution process was performed for wild type. As for the variants, only the reconstituted wild type exhibited a discrete band after native PAGE (Fig. 2). There was intermittent success and low yields in the reconstitution of both C59X and C180X (X = A or S) indicating they were unstable.

96

Figure 2. Coomassie blue stained native PAGE of wild type Isf and variants. Top panel contains as-purified wild type Isf (25 µg), reconstituted Isf (25 µg), and cysteine to alanine variants (25 µg; except 18 µg for 180A). Bottom panel contains as-purified wild type Isf (18 µg), reconstituted cysteine to serine variants (25 µg for C16S and C50S, 18 µg for C47S, C53S, and C180S).

97

Since the proteins showed less precipitation during refolding in the presence of 0.5 M arginine, cofactors reconstitution in C59X and C180X in the presence of arginine were performed. In some instances with no consistency, this method yields higher recovery of reconstituted proteins, which is indicated in higher intensity of EPR signal (Fig. 8).

Spectroscopic characterization of reconstituted wild type Isf and variants. The UV-visible spectra of denatured and renatured wild type Isf showed no absorbance characteristic of either iron-sulfur clusters or FMN (Fig 3) suggesting the complete loss of both redox components. The UV-visible spectrum of the reconstituted Isf was nearly identical to the as-purified wild type suggesting the reconstituted protein might have properties similar to as-purified Isf. The UV-visible absorption spectra for the reconstituted variants were similar to the wild type (Fig. 4, 5); however, the ratio of FMN absorbance at 378 nm relative to iron-sulfur cluster absorbance centered at 430 nm was generally lower for all except C16A and C16S. These results suggest significantly lower incorporation of FMN relative to iron-sulfur clusters for all variants except C16A and C16S. The UV-visible spectrum of C59S contained no features characteristic of ironsulfur cluster incorporation, a result that was confirmed by EPR (data not shown).

EPR spectrum of wild type Isf, alanine and serine variants were recorded and compared to determine cysteines that participate in ligating iron-sulfur cluster in Isf. The reduced as-purified Isf exhibited an EPR spectrum with g-values of 2.06, 2.03, 1.92, 1.86, 1.81 (Fig. 6, Table 1), results that are nearly identical to a previous report (2) in which the authors attributed the complexity of the spectrum to heterogeneity of the sample (refer to chapter 3). We were able to distinguish two distinct species based on power and temperature dependencies, one with g values of 2.06, 1.92, and 1.81 and another with g values of 2.03, 1.92, and 1.86 (Fig. 6). The ratio of these species varied in different Isf preparations suggesting the as-purified protein exists in two distinct conformational states. Incubation of as purified Isf with ethylenediaminetetraacetic acid sodium salt (EDTA) resulted in nearly complete destruction of 4Fe-4S center as followed by EPR (Fig. 6). However, the overwhelming majority of the center reconstituted to the g = 2.03, 1.92, and 1.81 species. This implies that under experimental conditions, this Isf

98

as-purified

reconstituted

0.1 A

renatured

denatured

280

330

380

430

480

530

Wavelength (nm)

Figure 3. UV-visible absorption spectra of as-purified, denatured, and reconstituted wild type Isf. The denatured protein was in 50 mM Tris-Cl (pH 7.6) containing 6 M guanidine-HCl. The as purified, renatured, and reconstituted proteins were in 50 mM Tris-Cl (pH 7.6) containing 200 mM NaCl. The spectra were recorded at 21o C. The amount of protein used for as-purified Isf was 80 µg, all other were 150 µg.

99

Isf

C16A C47A

C50A C53A

0.2 A

C59A

C180A

280

330

380

430

480

530

Wavelength (nm)

Figure 4. UV-visible absorption spectra of wild type Isf and alanine variants. The samples (500 µl) were in 50 mM Tris-Cl (pH 7.6) containing 200 mM NaCl, and the spectra were recorded at 21oC. The amount of protein used for wild type Isf was 80 µg; all variants were 120 µg.

100

Isf C16S

0.2 A

C47S

C50S

C53S

280

330

380

430

480

530

Wavelength (nm)

Figure 5. UV-visible absorption spectra of wild type Isf and serine variants. The samples (500 µl) were in 50 mM Tris-Cl (pH 7.6) containing 200 mM NaCl, and the spectra were recorded at 21oC. The amount of protein used for wild type Isf was 80 µg; all variants were 120 µg.

101

Figure 6. EPR spectra of reduced wild type and reconstituted Isf. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Proteins were reduced by sodium dithionite.

102

conformation is energetically more favorable and it could be achieved more easily. Reconstitution of wild type Isf that had been denatured and renatured also exhibited a 4Fe-4S EPR spectrum identical to wild type with evidence for both species. This result indicated that the procedure for reconstituting the Isf apoprotein yielded a 4Fe-4S cluster with an environment identical to the wild type.

The reduced C16A and C16S variants exhibited EPR spectra with g-values of 2.06, 2.04, 1.92, 1.86, 1.82 (Fig. 7, Table 1) characteristic of wild type Isf with two distinct 4Fe-4S species. The reduced C180A and C180S variants showed EPR spectra (Fig. 8, 9, Table 1) with line shapes and g-values (2.07, 2.04, 1.93, 1.86, 1.81) nearly identical to one of the 4Fe-4S species present in the wild-type Isf spectrum. A minor contribution of the other species was also observed as a shoulder at g 2.07. These results strongly indicate that C16 and C180 do not participate in ligation of the 4Fe-4S cluster of Isf. This conclusion is further supported by sequence comparisons showing that C16 and C180 are not conserved with putative Isf proteins (Fig. 1). The predominance of one 4Fe-4S species in the EPR spectra of the C180 variants suggests one of the conformations possible for wild type Isf is preferred in these variants. This proposal is consistent with the instability of the C180A and C180S variants which suggests that C180 is important for the overall structural integrity of the protein.

Cysteines 47, 50, 53 and 59 are strictly conserved among putative Isf sequences (Fig. 1) as a compact motif CX2CX2CX4-7C suggesting the motif is essential and, therefore, a candidate for ligation of the 4Fe-4S cluster. EPR spectroscopy of the reduced C50A or C59A variants detected no [4Fe-4S]1+ cluster; however, spectra of the oxidized variants had linewidths and g-values typical for [3Fe-4S]2+ clusters (Fig. 10, 11, Table 1). These results strongly indicate that C50 and C59 are involved in ligation of the 4Fe-4S cluster in Isf. The results also show that other ligands cannot substitute for C50 and C59 to preserve the 4Fe-4S cluster in these variants. A [4Fe-4S]1+ cluster was detected in both of the reduced C47A and C53A variants by EPR spectroscopy; however, there were significant differences in the line widths and g-values between the spectra of the two

103

g =

C16A/S C16S C16A

2.3

2.2

2.1

2.0 g -value

1.9

1.8

Figure 7. EPR spectra of reduced C16X (X = A or S). EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Proteins were reduced by sodium dithionite.

104

g =

C180A +Arg -Arg

2.3

2.2

2.1

2.0 g -value

1.9

1.8

Figure 8. EPR spectra of reduced C180A. (+ Arg) The protein was denatured, refolded as described in experimental procedures. However, the reconstitution process was performed immediately after refolding with no dialysis to remove arginine. (­Arg) The protein was denatured, refolded, dialyzed to remove arginine, and then reconstituted. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Proteins were reduced by sodium dithionite.

105

g =

C180S

2.3

2.2

2.1 g -value

2.0

1.9

1.8

Figure 9. EPR spectrum of reduced C180S. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein was reduced by sodium dithionite.

106

g =

C50A

2.05

2.00

1.95 g -value

1.90

1.85

Figure 10. EPR spectrum of as-purified C50A. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT.

107

g =

C59A

2.2

2.1

2.0 g -value

1.9

Figure 11. EPR spectra of as-purified C59A. EPR conditions were as follows: temperature 15 K, microwave power 1.26 mW, modulation amplitude 1 mT.

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variants. The line widths and g-values for both variants were also significantly different from the spectra of either of the two species present in the reduced form of as-isolated wild-type Isf (Fig. 12, 13, Table 1). A low-intensity [3Fe-4S]2+ EPR signal was detected in the oxidized C47A and C53A variants. These data strongly indicate that one or more ligands to the 4Fe-4S cluster changed in these variants, a result suggesting that C47 and C53 are ligands to the 4Fe-4S cluster in wild type Isf. The EPR results obtained for the C47A, C50A, C53A, and C59A variants, combined with strict conservation of the CX2CX2CX4-7C motif in putative Isf sequences from diverse species (Fig. 1), strongly suggest involvement of the motif in ligation of the 4Fe-4S cluster in Isf.

Although other residues could replace cysteine as a ligand to the 4Fe-4S clusters in C47A and C53A, we consider it most likely that 2-mercaptoethanol is an external thiolate ligand in these variants for the following reasons. The buffer used for reconstitution of the variants contained 2-mercaptoethanol which has been shown to serve as an external ligand to the 4Fe-4S cluster in the C51D and C14G variants of PsaC from Photosystem I in Synechocystis sp. PCC 6803 (11, 28). The reconstitution conditions used in this work were nearly identical to the conditions used to reconstitute iron-sulfur clusters in PsaC. Thiolate ligands in C53A and C47A are expected to occupy different positions in the coordination sphere of the 4Fe-4S cluster consistent with differences in the EPR spectra recorded for these variants. Thiolate ligation is also consistent with differences in the EPR signals of the C53A and C47A variants compared to wild-type Isf. The EPR spectra of the reduced C47S, C50S, and C53S variants indicated the presence of 4Fe-4S clusters (Fig. 14-16, Table 1). The EPR spectra of C47S, C50S and C53S suggest the presence of two different ligand species. These features could potentially derive from the use of substituted serine and 2-mercaptoethanol presence in the reconstitution system. The role of introduced serine residues in ligation of iron-sulfur clusters has been determined for several proteins by substitution with alanine in which case the native cluster does not assemble if the replaced cysteine or serine residues are

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g =

C47A

2.3

2.2

2.1

2.0 g -value

1.9

1.8

Figure 12. EPR spectrum of reduced C47A. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein was reduced by sodium dithionite.

110

g =

C53A

2.3

2.2

2.1

2.0 g -value

1.9

1.8

Figure 13. EPR spectrum of reduced C53A. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein was reduced by sodium dithionite.

111

g =

C47S

2.3

2.2

2.1 g -value

2.0

1.9

1.8

Figure 14. EPR spectrum of reduced C47S. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein was reduced by sodium dithionite.

112

g =

C50S

2.3

2.2

2.1 g -value

2.0

1.9

1.8

Figure 15. EPR spectrum of reduced C50S. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. The protein was reduced by sodium dithionite.

113

g =

C53S

2.3

2.2

2.1 g -value

2.0

1.9

1.8

Figure 16. EPR spectrum of reduced C53S. EPR conditions were as follows: temperature 15 K, microwave power 20 mW, modulation amplitude 1 mT. Protein was reduced by sodium dithionite.

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Table 1. EPR properties of wild-type Isf and variants.

Protein As-purified wild-type

g values 2.06, 1.92, 1.81 2.03, 1.92, 1.86

Iron-sulfur center type [4Fe-4S]

Reconstituted wild-type

2.06, 1.92, 1.81 2.03, 1.92, 1.86

[4Fe-4S]

C16X X = A or S C47A C47S C50A C50S C53A C53S C59A C180X X = A or S

2.06, 1.92, 1.82 2.04, 1.92, 1.86 2.05, 1.93, 1.89 2.05, 1.94, 1.86, 1.80 2.01,1.99 2. 07, 2.04,1.92, 1.90,1.82 2.03,1.91,1.89 2.05, 1.99, 1.93, 1.91, 1.82 2.01,1.99 2.04, 1.93, 1.86 With minor contribution from 2.07, 1.93, 1.81

[4Fe-4S]

[4Fe-4S] [4Fe-4S] [3Fe-4S] [4Fe-4S] [4Fe-4S] [4Fe-4S] [3Fe-4S] [4Fe-4S]

115

required ligands (16, 17). Thus, conversion of the 3Fe-4S cluster of C50A to a 4Fe-4S cluster by replacement with serine suggests that serine can substitute for C50 in Isf.

Functional characterization of Isf variants. It has been proposed that electron flow is from reduced ferredoxin A to the 4Fe-4S cluster of Isf and then to FMN based only on midpoint potential values (2); thus, the ability of ferredoxin A to reduce FMN in the stable variants was investigated to test this hypothesis (Table 2). The reduction of FMN was followed at A476 to avoid interference due to reduction of the iron-sulfur clusters. The as- purified and reconstituted Isf were reduced at similar rates (Table 2) consistent with the spectroscopic characterizations indicating that the reincorporated 4Fe4S and FMN were functionally similar to as-purified wild-type Isf. The C47, C50, and C53 variants showed either several-fold lower or higher rates compared to wild type (table 2), a result which suggests that the 4Fe-4S cluster is required to transfer electrons from ferredoxin A to FMN. This result is consistent with results suggesting that the reconstitution of FMN into apo-protein is diminished by substitution of residues in the motif (C47, C50, C53, and C59) coordinating the 4Fe-4S cluster. Local conformational changes in the environment of the 4Fe-4S cluster could potentially influence the reconstitution of FMN if it were adjacent to the 4Fe-4S cluster for electron transfer.

The FMN reduction in C47A and C50S was decreased relative to wild type demonstrating that the 4Fe-4S clusters in these variants are able to function, albeit less effectively, with the ligands that replaced C47 and C50. The several-fold higher rates relative to wild type Isf for variants C53A and C47S is unexplained; however, the results clearly indicate that the 4Fe-4S clusters in these variants are fully functional suggesting that the ligands replacing C53 and C47 conserve essential properties of the wild type 4Fe-4S cluster. Midpoint potentials of 3Fe-4S clusters are generally less negative than 4Fe-4S clusters, the Em range for 3Fe-4S is +80 to ­ 420 mV while the 4Fe-4S center is +80 to ­ 700 mV. Thus, it is hypothesized that the lower rate of FMN reduction exhibited by C50A could possibly be influenced by the redox potential of the 3Fe-4S cluster in this variant similar to that predicted for the 4Fe-4S cluster in the corrinoid/iron-

116

Table 2. Rates for reduction of FMN in wild type Isf and variants. Rate a 0.52 ± 0.03 0.59 ± 0.08 0.27 ± 0.01 0.14 ± < 0.01 0.14 ± < 0.01 1.53 ± 0.01 0.49 ± < 0.01 1.38 ± 0.26 0.21 ± < 0.01 0.50 ± 0.09

Protein As-purified wild type Reconstituted wild type C16A C47A C50A C53A C16S C47S C50S C53S

cluster type 4Fe-4S 4Fe-4S 4Fe-4S 4Fe-4S 3Fe-4S 4Fe-4S 4Fe-4S 4Fe-4S 4Fe-4S 4Fe-4S

a:

Change in absorbance at 476 nm/min/µmole FMN with ferredoxin A as the electron

donor (see Materials and Methods).

117

sulfur protein from C. thermoaceticum (15). Although the results suggest C16 is not involved in ligation of the 4Fe-4S cluster, reduction of FMN in C16A was impaired suggesting this residue indirectly influences the transfer of electrons from ferredoxin to FMN.

DISCUSSION

The data presented here provides biochemical confirmation of a novel and unusually compact motif (CX2CX2CX4-7C) for ligation of the 4Fe-4S cluster. Sequence comparisons (Fig. 1) suggest that the spacing between the first and the second cysteines in this motif is rigid while spacing between the third and the fourth cysteines is somewhat variable; however, additional site-directed mutagenesis experiments are necessary to test this hypothesis. This motif differs from those in ferredoxins (typically CX2CX2C and a distal C) and high potential iron proteins (typically CX2CX16CX13C) where the ligands to the 4Fe-4S clusters are more dispersed in the primary structure. Other examples of highly compact motifs ligating a 4Fe-4S cluster are endonuclease III and MutY (CX6CX2CX5C); however, these clusters are resistant to oxidation and reduction (19, 20) and function instead to position basic residues for interaction with the phosphate backbone of DNA. Another compact cysteine motif (CX2CX11CX5C) ligating a redox resistant 4Fe-4S cluster is present in the LRR protein for which the physiological function is unknown (18). Still other novel regulatory and enzymatic roles have been described for iron-sulfur clusters (3, 10). The results reported here are consistent with a role for the 4Fe-4S cluster in transfer of electrons from the physiological electron donor (ferredoxin A) to FMN; however, alternate or additional roles cannot be ruled out.

This investigation has also provided insight into iron-sulfur cluster and FMN selfassembly in Isf in vivo. The present work showed that Isf variants could be refolded in the presence of arginine to a conformational state such that the variant could be reconstituted by treating the protein with ferric ion, sulfide, and FMN. It is proposed that arginine helps to reshuffle molecules trapped in non-productive reactions, which results in increased refolding efficiency (4). Unfortunately, we were unable to identify

118

conditions to produce an appreciable yield of the reconstituted C59X and C180X (X = A or S) variants. Refolding in the presence of ferric ion, sulfide, and FMN was performed for these variants; however, the majority of the protein precipitated during concentration and buffer exchange. Nevertheless, we were successful in characterizing these variant spectroscopically (see Results). Replacement of cysteines in the proposed 4Fe-4S binding motif (C47, C50, C53, and C59) suggests that integrity of the 4Fe-4S cluster is important for reconstitution of FMN. Changes in these residues resulted in poor incorporation of FMN relative to the iron-sulfur cluster.

The results presented here show that the biochemical and physiological properties of the iron-sulfur cluster are remarkably stable to changes in ligation at the same time it is very sensitive to the ligand environment. All of the serine variants contained a 4Fe-4S cluster, except C59S and all of the alanine variants contained an iron-sulfur cluster, two of which were of the 3Fe-4S type. Furthermore, the FMN of all the variants was reduced by ferredoxin A at a significant rate compared to wild type, a result demonstrating that the iron-sulfur clusters were competent in transferring electrons from ferredoxin A to FMN. This resiliency of the 4Fe-4S cluster to changes in the ligation environment may be a consequence of the unusually compact nature of the motif coordinating the cluster.

Examples of 4Fe-4S clusters bridging between protein subunits have previously been shown for the nitrogenase Fe-protein and the Fx cluster in photosystem I. The possibility that the cysteines of the motif which ligate the 4Fe-4S centers are shared between subunits can not be completely ruled out based on available experimental data. However, we see no evidence of spin coupling between two clusters, although that effect is distance dependent.

The N-terminal half of the deduced sequence of Isf contains regions (Fig. 1, underlined residues) with identity to the flavin-binding domain of flavodoxins (13). The unusually compact nature of the cysteine motif coordinating the 4Fe-4S cluster obviates the need for a remote cysteine suggesting the possibility that Isf could have evolved by insertion of a small ancestral 4Fe-4S protein, containing the compact motif,

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into the N-terminal half of an ancestral flavodoxin. A search of the databases revealed no additional sequences with significant identity to residues 23-85 in the M. thermophila Isf sequence (Fig. 1) that includes the cysteine motif.

In addition to the motif ligating the 4Fe-4S cluster, two other cysteines (C16 and C180) are present in the Isf sequence (Fig. 1). Although the evidence suggests C16 and C180 are not involved in coordination of the 4Fe-4S cluster, the C16A variant was impaired in the ability to catalyze ferredoxin A-dependent reduction of FMN. A direct role for C16 in electron transfer was ruled out by the observation that C16A is partially, and C16S fully, competent in electron transfer from ferredoxin A to FMN compared with wild type Isf. Sequence comparisons (Fig. 1) indicate that a threonine residue in Isf homologs may replace C16 of the M. thermophila Isf. This finding suggests that a hydroxyl or sulfhydryl group is important for maintaining a conformation required for interaction of ferredoxin A with the 4Fe-4S cluster of Isf or intramolecular electron transfer from the cluster to FMN. The results obtained for C180 suggest this residue is not involved in iron-sulfur cluster ligation, however, instability of the C180X (X = A or S) variants suggests that this cysteine is required only for the overall structural integrity of the protein.

ACKNOWLEDGEMENTS We would like to thank M.L. Antokine and Dr. J.H. Golbeck at Pennsylvania State University for performing the EPR spectroscopy. We thank Dr. R.C. Thauer for suggestion on the arginine refolding method and a special thank you to R.D. Miles for her critical reading of the manuscript. Work described here was partially supported by the Department of Energy Basic Energy Sciences Grant No. DE-FG02-95ER20198 (JGF) and MCB-9723661 (JHG). UL was supported by MOSTE grant from Thailand.

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CHAPTER 6 SUMMARY AND FURTURE DIRECTIONS Advances in understanding the biochemical reactions involved in carbon transformations during methanogenesis have inspired investigations of how electrons are transported to generate energy for the cell. The characterization of a novel redox protein (Isf) in this study provides a better understanding of electron transport in the acetate fermentation pathway. Information derived from the sequence of the heterologously produced iron-sulfur flavoprotein (Isf) from the archaeon Methanosarcina thermophila shows many striking features distinct from any known iron-sulfur proteins or flavoproteins. The sequence reveals a novel cysteine motif that has been shown by site-directed mutagenesis and spectroscopic analyses to accommodate a 4Fe-4S center. An unusual higher stability of hydroquinone relative to semiquinone prompts an investigation of the environment in the FMN binding site that results in hydroquinone formation. The three-dimensional structure of this archaeal Isf is being solved using X-ray crystallography in collaboration with Dr. C. Bremnane and Prof. D. Rees at Caltech. Since Isf of M. thermophila is the prototype for this protein family and there is no significant sequence identity with known proteins, the structure may help to elucidate the environment of redox centers. The structure may support the proposed functions of these two cofactors by showing if both of them are able to participate in electron transfer as expected. This will lead to a better understanding of the mechanism for electron transfer. The heterologously produced Isf has been studied, but the native Isf from M. thermophila has not yet been purified. Thus, it is not possible to propose with confidence that they share the same properties. Attempts to identify Isf from M. thermophila using Western blots were not successful. This protein may be expressed only at very low levels and only under certain growth conditions. Northern blot analysis is an alternative approach to examine the expression and regulation of the isf gene.

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Although the spectroscopic techniques and site-directed mutagenesis reported here provide structural information for Isf, the function of Isf remains an issue. The involvement of Isf in electron transport CO-dependent CoM-S-S-CoB reduction has been shown, and there is evidence that ferredoxin A is a direct electron donor for Isf. In contrast, the physiological oxidative partner is not known. Passing M. thermophila cell extract through an affinity column to which Isf is bound may be used to identify proteins that interact with Isf and possibly function as its electron acceptor. Biochemical characterization of Isf-like sequences from other metabolically diverse microbes may reveal their roles in these microbes. These sequences may provide an opportunity to study evolutionary convergence of Isf.

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Curriculum Vista UBOLSREE LEARTSAKULPANICH Department of Biochemistry and Anaerobic Microbiology Virginia Polytechnic Institute and State University Blacksburg, VA 24061 204 S.Frear Bld. BMB, Penn State University University Park PA 16802-4500 814-863-5822 [email protected] PERSONAL Date of birth: 2/10/71 (Bangkok, Thailand) Place of birth: Bangkok, Thailand EDUCATION: Aug 1993 - present: Doctoral candidate, Department of Biochemistry and Anaerobic Microbiology Virginia Polytechnic Institute and State University (VPI&SU), Blacksburg, VA. Aug 1995 - present: Department of Biochemistry and Molecular Biology Pennsylvania State University, University Park, PA. In absentia from Virginia Polytechnic Institute and State University Apr 1992 ­ 1993 MS. candidate, Department of Biochemistry, Mahidol University, Bangkok, Thailand Jun 1988 - 1992 B.S. Biochemistry, Chulalongkorn University, Bangkok, Thailand PROFESSIONAL EXPERIENCE Jun 1994 - Aug 1994: Graduate Teaching Assistant, Department of Biochemistry and Anaerobic Microbiology. VPI&SU. Laboratory instructor for BAM5104 Advanced Methods of Biochemical Analysis Apr 1991 ­ May 1991: Student training at Chareonpokapun group (CP), Samut Prakarn, Thailand HONORS AND AWARDS: Royal Thai Scholarship for Ministry of Science, Technology and Energy (MOSTE) 447 W. Clinton Ave., # 408 State College PA 16801 814-861-6251

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National Science and Development Agent scholarship (NSDA) The first rang student of the 1992 MS. candidate class, Mahidol University The first range student for 1992 MS. entrance examination, Mahidol Unversity BS. with Second class honor degree, Chulalongkorn University PUBLICATIONS: Becker D.F., Leartsakulpanich U., Surerus K.K., Ferry J.G., and S.W. Ragsdale. 1998. Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila. J. Biol Chem. 273:26462-26469 Leartsakulpanich U, Antokine M.L., Golbeck J.H., and J.G Ferry. A novel [4Fe4S] iron-sulfur cluster binding motif in the iron-sulfur flavoprotein of Methanosarcina thermophila (in preparation) ABSTRACTS AND PRESENTATIONS: Inorganic Biology Summer Workshop (IBSW 98), Athens, GA Jul 25 ­ Aug 5, 1998 Leartsakulpanich U, Antokine M.L., Golbeck J.H., and J.G. Ferry. 1998. Ligands to the 4Fe-4S center in the iron-sulfur flavoprotein from Methanosarcina thermophila and proposed physiological function Penn State Sixteenth Summer Symposium in Molecular Biology Microbial Structural Biology: Novel Enzymes from Diverse Microbes Aug 7-9, 1997 Leartsakulpanich U., Becker D.F., Ragsdale S.W., Borup B., Aldrich H.C., and J.G.Ferry. Characterization of the iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila.

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