Hydrogenases (H2ases) catalyze the reversible oxidation of molecular hydrogen and play a central role in microbial energy metabolism. Most of these enzymes are found in Archaea and Bacteria, but a few are present in Eucarya as well. They can be distributed into three classes: the [Fe]-H2ases, the [NiFe]-H2ases, and the metal-free H2ases. The vast majority of known H2ases belong to the first two classes, and over 100 of these enzymes have been characterized genetically and/or biochemically. Compelling evidence from sequences and structures indicates that the [NiFe]- and [Fe]-H2ases are phylogenetically distinct classes of proteins. The catalytic core of the [NiFe]-H2ases is a heterodimeric protein, although additional subunits are present in many of these enzymes. Functional classes of [NiFe]-H2ases have been defined, and they are consistent with categories defined by sequence similarity of the catalytic subunits. The catalytic core of the [Fe]-H2ases is a ca. 350-residue domain that accommodates the active site (H-cluster). A few monomeric [Fe]-H2ases are barely larger than the H-cluster domain. Many others are monomeric as well, but possess additional domains that contain redox centers, mostly iron–sulfur. Some [Fe]-H2ases are oligomeric. The modular structure of H2ases is strikingly illustrated in recently unveiled sequences and structures. It is also remarkable that most of the accessory domains and subunits of H2ases have counterparts in other redox complexes, in particular NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. Microbial genome sequences are bringing forth a significant body of additional H2ase sequence data and contribute to the understanding of H2ase distribution and evolution. Altogether, the available data suggest that [Fe]-H2ases are restricted to Bacteria and Eucarya, while [NiFe]-H2ases, with one possible exception, seem to be present only in Archaea and Bacteria. H2ase processing and maturation involve the products of several genes which have been identified and are currently being characterized in the case of the [NiFe]-H2ases. In contrast, near to nothing is known regarding the maturation of the [Fe]-H2ases. Inspection of the currently available genome sequences suggests that the [NiFe]-H2ase maturation proteins have no similar counterparts in the genomes of organisms possessing [Fe]-H2ases only. This observation, if confirmed, would be consistent with the phylogenetic distinctiveness of the two classes of H2ases. Sequence alignments of catalytic subunits of H2ases have been implemented to construct phylogenetic trees that were found to be consistent, in the main, with trees derived from other data. On the basis of the comparisons performed and discussed here, proposals are made to simplify and rationalize the nomenclature of H2ase-encoding genes.

Classification and phylogeny of hydrogenases.

Methanogenic archaea have an unusual type of metabolism because they use H2 + CO2, formate, methylated C1 compounds, or acetate as energy and carbon sources for growth. The methanogens produce methane as the major end product of their metabolism in a unique energy-generating process. The organisms received much attention because they catalyze the terminal step in the anaerobic breakdown of organic matter under sulfate-limiting conditions and are essential for both the recycling of carbon compounds and the maintenance of the global carbon flux on Earth. Furthermore, methane is an important greenhouse gas that directly contributes to climate changes and global warming. Hence, the understanding of the biochemical processes leading to methane formation are of major interest. This review focuses on the metabolic pathways of methanogenesis that are rather unique and involve a number of unusual enzymes and coenzymes. It will be shown how the previously mentioned substrates are converted to CH4 via the CO2-reducing, methylotrophic, or aceticlastic pathway. All catabolic processes finally lead to the formation of a mixed disulfide from coenzyme M and coenzyme B that functions as an electron acceptor of certain anaerobic respiratory chains. Molecular hydrogen, reduced coenzyme F420, or reduced ferredoxin are used as electron donors. The redox reactions as catalyzed by the membrane-bound electron transport chains are coupled to proton translocation across the cytoplasmic membrane. The resulting electrochemical proton gradient is the driving force for ATP synthesis as catalyzed by an A1A0-type ATP synthase. Other energy-transducing enzymes involved in methanogenesis are the membrane-integral methyltransferase and the formylmethanofuran dehydrogenase complex. The former enzyme is a unique, reversible sodium ion pump that couples methyl-group transfer with the transport of Na+ across the membrane. The formylmethanofuran dehydrogenase is a reversible ion pump that catalyzes formylation and deformylation of methanofuran. Furthermore, the review addresses questions related to the biochemical and genetic characteristics of the energy-transducing enzymes and to the mechanisms of ion translocation.

The unique biochemistry of methanogenesis.

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2−1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni−Fe active site. The CO was not replaced with H2 in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni−C distances and Ni−C−O angles were about 1.77 A and 160°, respectively, except for one case (1.72 A and 135°), in which an additional electron density peak between the CO and Sγ(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sγ(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sγ(Cys546) atoms suggest that these two atoms at the Ni−Fe act...

Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen.

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U·mg protein−1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 µm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U·mg protein−1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 µm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 µm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

The membrane-associated coenzyme F420-reactive hydrogenase of the anaerobic methanogenic archaeon Methanosarcina barkeri Fusaro has been purified 95-fold to apparent homogeneity. A new purification procedure and altered storage conditions gave substantially higher yield (13.4% versus 4.3%) and specific coenzyme F420-reducing activity (82.8 μmol · min−1· mg protein−1 versus 11.5 μmol · min−1· mg protein−1) than reported previously [Fiebig, K. & Friedrich, B. (1989) Eur. J. Biochem. 184, 79–88]. The predominant coenzyme F420-reactive form of the hydrogenase has an apparent molecular mass of 198 kDa and is composed of three non-identical subunits with apparent molecular masses of 48 (α), 33 (β), and 30 kDa (γ), apparently in a stoichiometry of α2β2γ1. This minimal coenzyme F420-reducing hydrogenase formed aggregates with apparent molecular masses of approximately 845 kDa. 1 mol of the 198-kDa form of hydrogenase contained 2 mol FAD, 2 mol nickel, 28–32 mol non-heme iron, and 34 mol acid-labile sulfur; in addition, 0.2 mol selenium was detected. The isoelectric point was 5.30. The amino acid sequence PXXRXEGH, where X is any amino acid, was found to be conserved in the N-termini of the putative nickel-binding subunits of most [NiFe]- and [NiFe]Sehydrogenases of methanogenic Archaea and Bacteria. However, this motif was not detected in the protein sequences of [Fe]hydrogenases. Maximal coenzyme F420-reducing activity was obtained with reductively reactivated enzyme at 55°C in the pH range 6.5–7.25. The Km, values of the purified enzyme for H2 with coenzyme F420 or methylviologen as electron acceptor were extremely low, namely 3μM and 4μM. The catalytic efficiency coefficients (kcat/Km) for H2 with both reducible cosubstrates were high: 2.5×107M−1· s−1 with coenzyme F420 and 6.9×107 M−1· S−1 with methylviologen.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.727_3.x

Regina Michel

Papers

Biochemical characterization of the 8-hydroxy-5-deazaflavin-reactive hydrogenase from Methanosarcina barkeri Fusaro.