Localization of the F420-reducing hydrogenase in Methanococcus voltae cells by immuno-gold technique

The F₄₂₀-reducing hydrogenase of Methanococcus voltae, which takes part in the terminal reduction step of methanogenesis, was localized in situ in ultrathin sections. This result was obtained by the immuno-gold technique using a high titer antiserum raised against the purified enzyme. Its specifity for the hydrogenase was shown by Western blot analysis. The hydrogenase of M. voltae was found to be membrane-associated.Abbreviations ELISA Enzyme linked immuno sorbent assay - F₄₂₀ 8-hydroxy-5-deazaflavin     

Function of H2-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F420 reduction with H2

Summary In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F₄₂₀ (F₄₂₀) with H₂ are present: (1) the F₄₂₀-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N ⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H₂-forming methylenetetrahydromethanopterin dehydrogenase and F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F₄₂₀-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H₂-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F₄₂₀ with H₂ is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system. Received: 9 September 1997 / Accepted: 30 October 1997     

Effect of methanogenic substrates on coenzyme F420-dependent N5,N10-methylene-H4MPT dehydrogenase,N5,N10-methenyl-H4MPT cyclohydrolase and F420-reducing hydrogenase activities in Methanosarcina barkeri

We measured F₄₂₀-dependent N⁵,N¹⁰-methylenetetrahydro-methanopterin dehydrogenase, N⁵, N¹⁰-methenyltetrahydro-methanopterin cyclohydrolase, and F₄₂₀-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H₂–CO₂-, methanol-, and H₂–CO₂+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H₂–CO₂, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H₂–CO₂- or methanol-grown cells, acetate-or H₂–CO₂ + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO₂-reduction pathway operated in the reverse direction. 2. When steps from CO₂ to CH₃-S-CoM in the CO₂-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.Abbreviations MF methanofuran - H₄MPT 5,6,7,8-tetrahydromethanopterin - HS-HTP 7-mercaptoheptanoylthreonine phosphate - CoM-S-S-HTP heterodisulfide of HS-CoM and HS-HTP - F₄₂₀ coenzyme F₄₂₀ (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative) - H₂F₄₂₀ reduced coenzyme F₄₂₀ - HC⁺=H₄MPT N⁵,N¹⁰-methenyl-H₄MPT - H₂C=H₄MPT N⁵,N¹⁰-methylene-H₄MPT - H₃C=H₄MPT N⁵-methyl-H₄MPT - BES 2-bromoethanesulfonic acid     

Regulation and function of ferredoxin-linked versus cytochrome b-linked hydrogenase in electron transfer and energy metabolism of Methanosarcina barkeri MS

Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H₂:CO₂ at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F₄₂₀-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu²⁺ column. Hydrogenase II did not react with ferredoxin or F₄₂₀, whereas hydrogenase I coupled to both ferredoxin and F₄₂₀. A reconstituted soluble protein system composed of purified CO dehydrogenase, F₄₂₀-reactive hydrogenase I fraction, and ferredoxin produced H₂ from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H₂ consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H₂ consumption coupled to methanogenesis and ATP synthesis, respectively.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport of Methanosarcina mazei Gö1

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F₄₂₀H₂ dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F₄₂₀H₂ was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The K_m values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F₄₂₀-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.     

Evidence for rapid microscale bacterial redox cycling of iron in circumneutral environments

The potential for microscale bacterial Fe redox cycling was investigated in microcosms containing ferrihydrite-coated sand and a coculture of a lithotrophic Fe(II)-oxidizing bacterium (strain TW2) and a dissimilatory Fe(III)-reducing bacterium (Shewanella alga strain BrY). The Fe(II)-oxidizing organism was isolated from freshwater wetland surface sediments which are characterized by steep gradients of dissolved O₂ and high concentrations of dissolved and solid-phase Fe(II) within mm of the sediment–water interface, and which support comparable numbers (10⁵–10⁶ mL⁻¹) of culturable Fe(II)-oxidizing and Fe(III)-reducing reducing. The coculture systems showed minimal Fe(III) oxide accumulation at the sand-water interface, despite intensive O₂ input from the atmosphere and measurable dissolved O₂ to a depth of 2 mm below the sand–water interface. In contrast, a distinct layer of oxide precipitates formed in systems containing Fe(III)-reducing bacteria alone. Examination of materials from the cocultures by fluorescence in situ hybridization indicated close physical juxtapositioning of Fe(II)-oxidizing and Fe(III)-reducing bacteria in the upper few mm of sand. Our results indicate that Fe(II)-oxidizing bacteria have the potential to enhance the coupling of Fe(II) oxidation and Fe(III) reduction at redox interfaces, thereby promoting rapid microscale cycling of Fe. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of a CO: heterodisulfide oxidoreductase system from acetate-grown Methanosarcina thermophila.

During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A. P. Clements, R. H. White, and J. G. Ferry, Arch. Microbiol. 159:296-300, 1993). The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis. A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase. Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts. The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB. The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2. In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase. The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides. The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b.     

VhuD Facilitates Electron Flow from H2 or Formate to Heterodisulfide Reductase in Methanococcus maripaludis

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H₂ via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F₄₂₀-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth.     

Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H₂ oxidized min^–1 (mg protein)^–1 with benzyl viologen as electron acceptor and an apparent K _m value for H₂ of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S^2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)⁺. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H₂-dependent cytochrome b reduction at a rate of 0.18 nmol min^–1 (mg protein)^–1. Received: 7 September 1995 / Accepted: 4 December 1995     

Methyl-coenzyme M reductase and other enzymes involved in methanogenesis from CO2 and H2 in the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F₄₃₀ as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO₂ (apparent V_max at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N ⁵,N¹⁰-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming), 33 U/mg; N ⁵,N¹⁰-methylenetetrahydromethanopterin reductase (coenzyme F₄₂₀ dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F₄₂₀-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent K_m values for these enzymes and the effect of salts on their activities were determined.The coenzyme F₄₂₀ present in M. kandleri was identified as coenzyme F₄₂₀-2 with 2 -glutamyl residues.Abbreviations H–S-CoM coenzyme M - CH₃–S-CoM methylcoenzyme M - H–S-HTP 7-mercaptoheptanoylthreonine phosphate - MFR methanofuran - CHO-MFR formyl-MFR - H₄MPT tetrahydromethanopterin - CHO–H₄MPT N ⁵-formyl-H₄MPT - CH=H₄MPT⁺ N ⁵,N¹⁰-methenyl-H₄MPT - CH₂=H₄MPT N ⁵,N¹⁰-methylene-H₄MPT - CH₃–H₄MPT N ⁵-methyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀ - 1 U= 1 mol/min     

New Mode of Energy Metabolism in the Seventh Order of Methanogens as Revealed by Comparative Genome Analysis of “Candidatus Methanoplasma termitum”

The recently discovered seventh order of methanogens, the Methanomassiliicoccales (previously referred to as “Methanoplasmatales”), so far consists exclusively of obligately hydrogen-dependent methylotrophs. We sequenced the complete genome of “Candidatus Methanoplasma termitum” from a highly enriched culture obtained from the intestinal tract of termites and compared it with the previously published genomes of three other strains from the human gut, including the first isolate of the order. Like all other strains, “Ca. Methanoplasma termitum” lacks the entire pathway for CO₂ reduction to methyl coenzyme M and produces methane by hydrogen-dependent reduction of methanol or methylamines, which is consistent with additional physiological data. However, the shared absence of cytochromes and an energy-converting hydrogenase for the reoxidation of the ferredoxin produced by the soluble heterodisulfide reductase indicates that Methanomassiliicoccales employ a new mode of energy metabolism, which differs from that proposed for the obligately methylotrophic Methanosphaera stadtmanae. Instead, all strains possess a novel complex that is related to the F₄₂₀:methanophenazine oxidoreductase (Fpo) of Methanosarcinales but lacks an F₄₂₀-oxidizing module, resembling the apparently ferredoxin-dependent Fpo-like homolog in Methanosaeta thermophila. Since all Methanomassiliicoccales also lack the subunit E of the membrane-bound heterodisulfide reductase (HdrDE), we propose that the Fpo-like complex interacts directly with subunit D, forming an energy-converting ferredoxin:heterodisulfide oxidoreductase. The dual function of heterodisulfide in Methanomassiliicoccales, which serves both in electron bifurcation and as terminal acceptor in a membrane-associated redox process, may be a unique characteristic of the novel order.     

Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H₂ and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO₂ fixation. The following enzymes were found [values in parentheses = μmol min^–1 (mg protein)^–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F₄₂₀-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F₄₂₀ (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO₂ fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N₂O with NO as intermediate (0.1 μmol N₂O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity. Received: 23 August 1996 / Accepted: 6 November 1996     

Pathways of energy conservation in methanogenic archaea

Methanogenic archaea are strictly anaerobic organisms that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H₂+CO₂ and formate are converted to CH₄ via the CO₂-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. A limited number of methanogenic organisms utilize acetate by the aceticlastic pathway. Redox reactions involved in these processes are partly catalyzed by membrane-bound enzyme systems that generate or, in the case of endergonic reactions, use electrochemical ion gradients. The H₂:heterodisulfide oxidoreductase, the F₄₂₀H₂:heterodisulfide oxidoreductase and the CO:heterodisulfide oxidoreductase, are novel systems that generate a proton motive force by redox-potential-driven H⁺ translocation. The methyltetrahydromethanopterin:coenzyme M methyltransferase is a unique, reversible sodium ion pump that couples methyl transfer with the transport of Na⁺ across the cytoplasmic membrane. Formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation, of methanofuran. In summary, the pathways are coupled to the generation of an electrochemical sodium ion gradient and an electrochemical proton gradient. Both ion gradients are used directly for ATP synthesis via membrane integral ATP synthases. The function of the above-mentioned systems and their components in the metabolism of methanogens are described in detail.Abbreviations DCCD N,N dicyclohexylcarbodiimide - F ₄₂₀ (N-l-Lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8 didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - H ₄MPT Tetrahydromethanopterin - HS-CoM 2-Mercaptoethanesulfonate - HS-HTP 7-Mercaptoheptanoyl-O-phospho-l-threonine - MF Methanofuran - Ms Methanosarcina - Mc Methanococcus - Mb Methanobacterium - SF 6847 3,5-Di-tert-butyl-4-hydroxybenzylidene-malononitrile - Electrochemical sodium ion gradient - Electrochemical proton gradient     

Purification and properties of the H2-oxidizing (uptake) hydrogenase of the N2-fixing anaerobe Clostridium pasteurianum W5

Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H₂-oxidizing (uptake) hydrogenase. The H₂-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H₂ oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H₂ produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a M_r of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S⁼ contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H₂-production and H₂-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH₃-grown cells was much lower than that in N₂-fixing cells.     

Partial Purification and Characterization of Two Hydrogenases from the Extreme Thermophile Methanococcus jannaschii

F₄₂₀-nonreactive and F₄₂₀-reactive hydrogenases have been partially purified from Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F₄₂₀-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F₄₂₀-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F₄₂₀-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F₄₂₀-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F₄₂₀-nonreactive hydrogenase was maximal at 80°C but was still detectable at 103°C. The maximum activities of F₄₂₀-reactive hydrogenase for F₄₂₀ and methyl viologen were measured at 80 and 90°C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103°C. Moreover, the half-life of the F₄₂₀-nonreactive hydrogenase at 70°C was over 9 h, and that of the F₄₂₀-reactive enzyme was over 3 h.