Mechanistic studies of methane biogenesis by methyl-coenzyme M reductase: evidence that coenzyme B participates in cleaving the C-S bond of methyl-coenzyme M.

Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and presteady-state kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-state kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.

Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Characterization of the B12- and iron-sulfur-containing reductive dehalogenase from Desulfitobacterium chlororespirans.

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

Cryoreduction of methyl-coenzyme M reductase: EPR characterization of forms, MCR(ox1) and MCR (red1).

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methyl-coenzyme M (CH(3)S-CH(2)CH(2)SO(3)) from methane. The active site is a nickel tetrahydrocorphinoid cofactor, factor 430, which in inactive form contains EPR-silent Ni(II). Two such forms, denoted MCR(silent) and MCR(ox1)(-)(silent), were previously structurally characterized by X-ray crystallography. We describe here the cryoreduction of both of these MCR forms by gamma-irradiation at 77 K, which yields reduced protein maintaining the structure of the oxidized starting material. Cryoreduction of MCR(silent) yields an EPR signal that strongly resembles that of MCR(red1), the active form of MCR; and stepwise annealing to 260-270 K leads to formation of MCR(red1). Cryoreduction of MCR(ox1)(-)(silent) solutions shows that our preparative method for this state yields enzyme that contains two major forms. One behaves similarly to MCR(silent), as shown by the observation that both of these forms give essentially the same redlike EPR signals upon cryoreduction, both of which give MCR(red1) upon annealing. The other form is assigned to the crystallographically characterized MCR(ox1)(-)(silent) and directly gives MCR(ox1) upon cryoreduction. X-band spectra of these cryoreduced samples, and of conventionally prepared MCR(red1) and MCR(ox1), all show resolved hyperfine splitting from four equivalent nitrogen ligands with coupling constants in agreement with those determined in previous EPR studies and from (14)N ENDOR of MCR(red1) and MCR(ox1). These experiments have confirmed that all EPR-visible forms of MCR contain Ni(I) and for the first time generated in vitro the EPR-visible, enzymatically active MCR(red1) and the activate-able "ready" MCR(ox1) from "silent" precursors. Because the solution Ni(II) species we assign as MCR(ox1)(-)(silent) gives as its primary cryoreduction product the Ni(I) state MCR(ox1), previous crystallographic data on MCR(ox1)(-)(silent) allow us to identify the exogenous axial ligand in MCR(ox1) as the thiolate from CoM; the cryoreduction experiments further allow us to propose possible axial ligands in MCR(red1). The availability of model compounds for MCR(red1) and MCR(ox1) also is discussed.

Characterization of a three-component vanillate O-demethylase from Moorella thermoacetica.

The Moorella thermoacetica aromatic O-demethylase was characterized as an inducible three-component system with similarity to the methanogenic methanol, methylamine, and methanethiol methyltransferases and to the O-demethylase system from Acetobacterium dehalogenans. MtvB catalyzes methyl transfer from a phenylmethylether to the cobalt center of MtvC, a corrinoid protein. MtvA catalyzes transmethylation from MtvC to tetrahydrofolate, forming methyltetrahydrofolate. Cobalamin can substitute for MtvC.

Characterization of the intramolecular electron transfer pathway from 2-hydroxyphenazine to the heterodisulfide reductase from Methanosarcina thermophila.

Heterodisulfide reductase (HDR) is a component of the energy-conserving electron transfer system in methanogens. HDR catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M (CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from Methanosarcina thermophila contains two b-hemes and two [Fe(4)S(4)] clusters. The physiological electron donor for HDR appears to be methanophenazine (MPhen), a membrane-bound cofactor, which can be replaced by a water-soluble analog, 2-hydroxyphenazine (HPhen). This report describes the electron transfer pathway from reduced HPhen (HPhenH(2)) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH(2) with the following values: k(cat) = 74 s(-1) at 25 degrees C, K(m) (HPhenH(2)) = 92 microm, K(m) (CoB-S-S-CoM) = 144 microm. Rapid freeze-quench EPR and stopped-flow kinetic studies and inhibition experiments using CO and diphenylene iodonium indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH(2). Fe-S cluster disruption by mersalyl acid inhibits heme reduction by HPhenH(2), suggesting that a 4Fe cluster is the initial electron acceptor from HPhenH(2). We propose the following electron transfer pathway: HPhenH(2) to the high potential 4Fe cluster, to the low potential heme, and finally, to CoB-S-S-CoM.

Crystal structure of a methyltetrahydrofolate- and corrinoid-dependent methyltransferase.

BACKGROUND: Methyltetrahydrofolate, corrinoid iron-sulfur protein methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation. It transfers the N5-methyl group from methyltetrahydrofolate (CH3-H4folate) to a cob(I)amide center in another protein, the corrinoid iron-sulfur protein. MeTr is a member of a family of proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or -sarcino)pterin. We report the first structure of a protein in this family. RESULTS: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 A resolution using multiwavelength anomalous diffraction methods. The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold. The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity. This homology permitted the methyl-H4folate binding site to be modeled. The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the methyltransferases and dihydropteroate synthetases. The most significant structural difference between these enzymes is in a loop structure above the active site. It is quite open in MeTr, where it can be modeled as the cobalamin binding site. CONCLUSIONS: The MeTr structure consists of a TIM barrel that embeds methyl-H4folate and cobamide. All related methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site. The observed structure is consistent with either a 'front' (N5) or 'back' (C8a) side protonation of CH3-H4folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).

The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway.

Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO(2). The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood. Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO(2) fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules. The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase. The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V(max) = 1.6 unit/mg (k(cat) = 3.2 s(-1)), K(m)(Acetyl-CoA) = 9 micrometer, and K(m)(CO(2)) = 2 mm. The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured. The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo. Measurements of its k(cat)/K(m) values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions. On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis.

Evidence for intersubunit communication during acetyl-CoA cleavage by the multienzyme CO dehydrogenase/acetyl-CoA synthase complex from Methanosarcina thermophila. Evidence that the beta subunit catalyzes C-C and C-S bond cleavage.

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of alpha, beta, gamma, delta, and epsilon subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO(2), transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H(4)MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H(4)MPT. The alpha and epsilon subunits are required for CO oxidation. The gamma and delta subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the beta subunit. The isolated beta subunit contains significant amounts of nickel. When proteases truncate the beta subunit, causing the CODH/ACS complex to dissociate, the amount of intact beta subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the beta subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the beta subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.

Channeling of carbon monoxide during anaerobic carbon dioxide fixation.

Carbon monoxide is an intermediate in carbon dioxide fixation by diverse microbes that inhabit anaerobic environments including the human colon. These organisms fix CO(2) by the Wood-Ljungdahl pathway of acetyl-CoA biosynthesis. The bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) catalyzes several key steps in this pathway. CO(2) is reduced to CO at a nickel iron-sulfur cluster called cluster C located in the CODH subunit. Then, CO is condensed with a methyl group and coenzyme A at cluster A, another nickel iron-sulfur cluster in the ACS subunit. Spectroscopic studies indicate that clusters A and C are at least 10-15 A apart. To gain a better understanding of how CO production and utilization are coordinated, we have studied an isotopic exchange reaction between labeled CO(2) and the carbonyl group of acetyl-CoA with the CODH/ACS from Clostridium thermoaceticum. When solution CO is provided at saturating levels, only CO(2)-derived CO is incorporated into the carbonyl group of acetyl-CoA. Furthermore, when high levels of hemoglobin or myoglobin are added to remove CO from solution, there is only partial inhibition of the incorporation of CO(2)-derived CO into acetyl-CoA. These results provide strong evidence for the existence of a CO channel between cluster C in the CODH subunit and cluster A in the ACS subunit. The existence of such a channel would tightly couple CO production and utilization and help explain why high levels of this toxic gas do not escape into the environment. Instead, microbes sequester this energy-rich carbon source for metabolic reactions.

Nickel containing CO dehydrogenases and hydrogenases.

The two redox catalysts described here can generate very low potential electrons in one direction and perform chemically difficult reductions in the other. The chemical transformations occur at unusual metal clusters. Spectroscopic, crystallographic, and kinetic analyses are converging on answers to how the metals in these clusters are arranged and how they are involved in the chemical and redox steps. The first structure of CO dehydrogenase, which will appear in the next year, will help define a firm chemical basis for future mechanistic studies. In the immediate future, we hope to learn whether the hydride intermediate in hydrogenase or the carbonyl intermediate in CO dehydrogenase bind to the Ni or Fe subsites in these heterometallic clusters. Or perhaps could they be bridged to two metals? Inter- and intramolecular wires have been proposed that connect the catalytic redox machine to proximal redox centers leading eventually to the ultimate redox partners. Elucidating the pathways of electron flow is a priority for the future. There is evidence for molecular channels delivering substrates to the active sites of these enzymes. In the next few years, these channels will be better defined. The products of CO2 and proton reduction are passed to the active sites of other enzymes and, in the case of H2, even passed from one organism to another. In the future, the mechanism of gas transfer will be uncovered. General principles of how these redox reactions are catalyzed are becoming lucid as the reactions are modeled theoretically and experimentally. Proton and CO2 reduction and the generation of C-C bonds from simple precursors are important reactions in industry. H2 could be the clean fuel of the future. Hopefully, the knowledge gained from studies of hydrogenase, CO dehydrogenase, and acetyl-CoA synthase can be used to improve life on earth.

Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer.

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.

The role of an iron-sulfur cluster in an enzymatic methylation reaction. Methylation of CO dehydrogenase/acetyl-CoA synthase by the methylated corrinoid iron-sulfur protein.

This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an SN2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center.

Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum.

Nitrate has been shown to shunt the electron flow in Clostridium thermoaceticum from CO2 to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J. M. Fröstl, C. Seifritz, and H. L. Drake, J. Bacteriol. 178:4597-4603, 1996). Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway. The CO oxidation activity in crude extracts of nitrate (30 mM)-supplemented cultures was fivefold less than that of nitrate-free cultures, while the H2 oxidation activity was six- to sevenfold lower. The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis. Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures. Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%). Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged. Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level. At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered. We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min-1 mg-1) in crude extracts of C. thermoaceticum. Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins. CO2 also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity.

Electrochemical and spectroscopic properties of the iron-sulfur flavoprotein from Methanosarcina thermophila.

An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.

Nucleotide excision repair in the third kingdom.

Nucleotide excision repair, a general repair mechanism for removing DNA damage, is initiated by dual incisions bracketing the lesion. In procaryotes, the dual incisions result in excision of the damage in 12- to 13-nucleotide-long oligomers, and in eucaryotes they result in excision of the damage in the form of 24- to 32-nucleotide-long oligomers. We wished to find out if Archaea perform excision repair. Using cell extracts from Methanobacterium thermoautotrophicum, we found that this organism removes UV-induced (6-4) photoproducts in the form of 10- to 11-mers by incising the sixth to seventh phosphodiester bond 5' to the damage and the fourth phosphodiester bond 3' to the damage.

The F420H2:heterodisulfide oxidoreductase system from Methanosarcina species. 2-Hydroxyphenazine mediates electron transfer from F420H2 dehydrogenase to heterodisulfide reductase.

F420H2-dependent CoB-S-S-CoM reduction as catalyzed by the F420H2:heterodisulfide oxidoreductase from Methanosarcina strains was observed in a defined system containing purified F420H2 dehydrogenase from Methanosarcina mazei Gö1, 2-hydroxyphenazine and purified heterodisulfide reductase from Methanosarcina thermophila. The process could be divided into two partial reactions: (1) reducing equivalents from F420H2 were transferred to 2-hydroxyphenazine by the F420H2 dehydrogenase with a Vmax value of 12 U/mg protein; (2) reduced 2-hydroxyphenazine acted as electron donor for CoB-S-S-CoM reduction as catalyzed by the heterodisulfide reductase. The specific activity was 14-16 U/mg protein at 37 degrees C and 60-70 U/mg protein at 60 degrees C. The partial reactions could be combined in the presence of both enzymes. Under these conditions reduced 2-hydroxyphenazine was rapidly oxidized by the heterodisulfide reductase thereby producing the electron acceptor for the F420H2 dehydrogenase. Above a concentration of 50 microM of 2-hydroxyphenazine, the specific activity of the latter enzyme reached the Vmax value. When other phenazines or quinone derivatives were used as electron carriers, the activity of F420H2-dependent CoB-S-S-CoM reduction was much lower than the rate obtained with 2-hydroxyphenazine. Thus, this water-soluble analogue of methanophenazine best mimics the natural electron acceptor methanophenazine in aqueous systems.

Nickel biochemistry.

Significant advances have been made in the past year in our understanding of the structure, function, and mode of regulation and assembly of nickel-containing enzymes. The highlight of 1997 was the elucidation of the methyl-CoM reductase structure.