F420H2 Dehydrogenase Research Articles

Coupling methanogenesis with iron reduction by acetotrophic Methanosarcina mazei zm-15.

Application of ferric iron is conventionally considered to inhibit methanogenesis in anoxic environments. Here we show that Methanosarcina mazei zm-15, a strain isolated from the natural wetland of Tibetan plateau, is capable of Fe(III) reduction, which significantly promotes its growth and methanogenesis. We grew Ms. mazei zm-15 in a medium containing acetate supplemented with Fe(III) in ferric citrate or ferrihydrite and to some cultures anthraquinone-2,6-disulfonate (AQDS) was applied as an electron shuttle. The reduction of Fe(III) species occurred immediately. Ferric citrate was more readily reduced than ferrihydrite. The X-ray diffraction spectra analysis showed the formation of magnetite from ferrihydrite and amorphous reduced products from ferrihydrite plus AQDS. The analysis of protein contents revealed that Fe(III) reduction contributed 36%-46% of the cell growth. The growth yield, estimated as protein increment per acetate consumed for Fe(III) reduction, increased by 20- to 30-fold compared with methanogenesis, which is in consistence with the difference in free energy available by Fe(III) reduction relative to methanogenesis. We propose that the outer-surface multiheme c-type cytochrome predicted from Ms. mazei zm-15 genome serves as the terminal reductase with the energy-converting hydrogenase and F420 H2 dehydrogenase involved in electron transport chain for Fe(III) reduction. The findings shed a light to better understand the ecophysiology of Methanosarcina in anaerobic environments.

Electron and Proton Flux for Carbon Dioxide Reduction in Methanosarcina barkeri During Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) is important in diverse methanogenic environments, but how methanogens participate in DIET is poorly understood. Therefore, the transcriptome of Methanosarcina barkeri grown via DIET in co-culture with Geobacter metallireducens was compared with its transcriptome when grown via H2 interspecies transfer (HIT) with Pelobacter carbinolicus. Notably, transcripts for the F420H2 dehydrogenase, Fpo, and the heterodisulfide reductase, HdrABC, were more abundant during growth on DIET. A model for CO2 reduction was developed from these results in which electrons delivered to methanophenazine in the cell membrane are transferred to Fpo. The external proton gradient necessary to drive the otherwise thermodynamically unfavorable reverse electron transport for Fpo-catalyzed F420 reduction is derived from protons released from G. metallireducens metabolism. Reduced F420 is a direct electron donor in the carbon dioxide reduction pathway and also serves as the electron donor for the proposed HdrABC-catalyzed electron bifurcation reaction in which reduced ferredoxin (also required for carbon dioxide reduction) is generated with simultaneous reduction of CoM-S-S-CoB. Expression of genes for putative redox-active proteins predicted to be localized on the outer cell surface was higher during growth on DIET, but further analysis will be required to identify the electron transfer route to methanophenazine. The results indicate that the pathways for electron and proton flux for CO2 reduction during DIET are substantially different than for HIT and suggest that gene expression patterns may also be useful for determining whether Methanosarcina are directly accepting electrons from other extracellular electron donors, such as corroding metals or electrodes.

A Metagenomics-Based Metabolic Model of Nitrate-Dependent Anaerobic Oxidation of Methane by Methanoperedens-Like Archaea.

Methane oxidation is an important process to mitigate the emission of the greenhouse gas methane and further exacerbating of climate forcing. Both aerobic and anaerobic microorganisms have been reported to catalyze methane oxidation with only a few possible electron acceptors. Recently, new microorganisms were identified that could couple the oxidation of methane to nitrate or nitrite reduction. Here we investigated such an enrichment culture at the (meta) genomic level to establish a metabolic model of nitrate-driven anaerobic oxidation of methane (nitrate-AOM). Nitrate-AOM is catalyzed by an archaeon closely related to (reverse) methanogens that belongs to the ANME-2d clade, tentatively named Methanoperedens nitroreducens. Methane may be activated by methyl-CoM reductase and subsequently undergo full oxidation to carbon dioxide via reverse methanogenesis. All enzymes of this pathway were present and expressed in the investigated culture. The genome of the archaeal enrichment culture encoded a variety of enzymes involved in an electron transport chain similar to those found in Methanosarcina species with additional features not previously found in methane-converting archaea. Nitrate reduction to nitrite seems to be located in the pseudoperiplasm and may be catalyzed by an unusual Nar-like protein complex. A small part of the resulting nitrite is reduced to ammonium which may be catalyzed by a Nrf-type nitrite reductase. One of the key questions is how electrons from cytoplasmically located reverse methanogenesis reach the nitrate reductase in the pseudoperiplasm. Electron transport in M. nitroreducens probably involves cofactor F420 in the cytoplasm, quinones in the cytoplasmic membrane and cytochrome c in the pseudoperiplasm. The membrane-bound electron transport chain includes F420H2 dehydrogenase and an unusual Rieske/cytochrome b complex. Based on genome and transcriptome studies a tentative model of how central energy metabolism of nitrate-AOM could work is presented and discussed.

Evidence for the involvement of two heterodisulfide reductases in the energy-conserving system of Methanomassiliicoccus luminyensis.

Methanomassiliicoccus luminyensis was isolated from the human gut, and requires H2 and methanol or methylamines to produce methane. The organism lacks cytochromes, indicating that it cannot couple membrane-bound electron transfer reactions with extrusion of H(+) or Na(+) ions using known methanogenic pathways. Furthermore, M. luminyensis contains a soluble hydrogenase/heterodisulfide reductase complex (MvhAGD/HdrABC) as found in obligate hydrogenotrophic methanogens, but the energy-conserving methyltransferase (MtrA-H) is absent. Thus, the question arises as to how this species synthesizes ATP. We present evidence that M. luminyensis uses two types of heterodisulfide reductases (HdrABC and HdrD) in a novel process for energy conservation. Quantitative RT-PCR studies revealed that genes encoding these heterodisulfide reductases showed high expression levels. Other genes with high transcript abundance were fpoA (part of the operon encoding the 'headless' F420 H2 dehydrogenase) and atpB (part of the operon encoding the A1 Ao ATP synthase). High activities of the soluble heterodisulfide reductase HdrABC and the hydrogenase MvhADG were found in the cytoplasm of M. luminyensis. Also, heterologously produced HdrD was able to reduce CoM-S-S-CoB using reduced methylviologen as an electron donor. We propose that membrane-bound electron transfer is based on conversion of two molecules of methanol and concurrent formation of two molecules of the heterodisulfide CoM-S-S-CoB. First the HdrABC/MvhADG complex catalyzes the H2 -dependent reduction of CoM-S-S-CoB and formation of reduced ferredoxin. In a second cycle, reduced ferredoxin is oxidized by the 'headless' F420 H2 dehydrogenase, thereby translocating up to 4 H(+) across the membrane, and electrons are channeled to HdrD for reduction of the second heterodisulfide.

Methanotrophic archaea possessing diverging methane-oxidizing and electron-transporting pathways

Anaerobic oxidation of methane (AOM) is a crucial process limiting the flux of methane from marine environments to the atmosphere. The process is thought to be mediated by three groups of uncultivated methane-oxidizing archaea (ANME-1, 2 and 3). Although the responsible microbes have been intensively studied for more than a decade, central mechanistic details remain unresolved. On the basis of an integrated analysis of both environmental metatranscriptome and single-aggregate genome of a highly active AOM enrichment dominated by ANME-2a, we provide evidence for a complete and functioning AOM pathway in ANME-2a. All genes required for performing the seven steps of methanogenesis from CO2 were found present and actively expressed. Meanwhile, genes for energy conservation and electron transportation including those encoding F420H2 dehydrogenase (Fpo), the cytoplasmic and membrane-associated Coenzyme B-Coenzyme M heterodisulfide (CoB-S-SCoM) reductase (HdrABC, HdrDE), cytochrome C and the Rhodobacter nitrogen fixation (Rnf) complex were identified and expressed, whereas genes encoding for hydrogenases were absent. Thus, ANME-2a is likely performing AOM through a complete reversal of methanogenesis from CO2 reduction without involvement of canonical hydrogenase. ANME-2a is demonstrated to possess versatile electron transfer pathways that would provide the organism with more flexibility in substrate utilization and capacity for rapid adjustment to fluctuating environments. This work lays the foundation for understanding the environmental niche differentiation, physiology and evolution of different ANME subgroups.

Purification and properties of an F420H2 dehydrogenase from Methanosarcina mazei Gö1

Abstract The F420H2 dehydrogenase is part of the energy-conserving F420H2:heterodisulfide oxidoreductase system in Methanosarcina mazei Go1. The enzyme was purified 75-fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S2− and 7 mol Fe. The specific activity was 17 U mg protein−1 (apparent Vmax) using F420H2 as electron donor (Km=7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and tetramethyl-p-benzoquinone. The protein did not exhibit hydrogenase activity since F420 was not reduced by hydrogen and H2 production from F420H2 was not observed. In contrast to the F420H2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Go1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Go1 contains FAD.

The membrane-bound electron transport system of Methanosarcina species.

Members of the genus Methanosarcina are strictly anaerobic archaea that derive their metabolic energy from the conversion of a restricted number of substrates to methane. H2 + CO2 are converted to CH4 via the CO2-reducing pathway, while methanol and methylamines are metabolized by the methylotrophic pathway. Two novel electron transport systems are involved in the process of methanogenesis. Both systems are able to use a heterodisulfide as electron acceptor and either H2 or F420H2 as electron acceptors and generate a proton-motive force by redox potential-driven H(+)-translocation. The H2:heterodisulfide oxidoreductase is composed of an F420-nonreducing hydrogenase and the heterodisulfide reductase. The latter protein is also part of the F420H2:heterodisulfide oxidoreductase system. The second component of this system is referred to as F420H2 dehydrogenase. The archaeal protein is a homologue of complex I of the respiratory chain from bacteria and mitochondria. This review focuses on the biochemical and genetic characteristics of the three energy-transducing enzymes and on the mechanisms of ion translocation.

The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases.

The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei Gö1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei Gö1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei Gö1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei Gö1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine.

Membrane-bound F 420 H 2 -dependent heterodisulfide reduction in Methanococcus voltae

Washed membranes prepared from H2+CO2- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F420H2 and the reduction of the heterodisulfide (CoB-S-S-CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F420H2 + COB-S-S-CoM --> F420 + CoM-SH + CoB-SH. These findings indicate that the reaction depends on a membrane-bound F420H2-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F420H2-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F420H2 oxidation is catalyzed by a membrane-associated F420-reducing hydrogenase. The purified protein catalyzed the H2-dependent reduction of methyl viologen and F420. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F420-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F420-reducing hydrogenase from Mc. voltae. A distinct F420H2 dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.

Inhibition of membrane-bound electron transport of the methanogenic archaeon Methanosarcina mazei Gö1 by diphenyleneiodonium.

The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Gö1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI.mg-1 protein and 45 nmol DPI.mg-1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2-hydroxyphenazine-dependent reactions as catalyzed by F420-nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2-dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane-bound cytochromes indicated that DPI inhibited CoB-S-S-CoM-dependent oxidation of reduced cytochromes and H2-dependent cytochrome reduction. Membrane-bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2-hydroxyphenazine were used as electron acceptors. Detailed examination of 2-hydroxy-phenazine-dependent F420H2-oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2-hydroxyphenazine, which increased from 35 microm to 100 microm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2-hydroxyphenazine to cytochrome b2 as part of the heterodisulfide reductase and from H2 to cytochrome b1 as subunit of the membrane-bound hydrogenase is affected in the presence of DPI. In case of the F420H2 dehydrogenase electron transport from FAD or from FeS centers to 2-hydroxyphenazine is inhibited.