Hydrogenase

There are three classes of CO dehydrogenase the NioCO dehydrogenase, the N16CO dehydrogenase/acetyl-CoA synthase, and the M06CO dehydrogenase. I will focus only on the first two enzyme classes in this review. 

Marjory Stephenson and Leonard Stickland named the enzyme in 1931 (Stephenson and Stickland, 1931). There are five types of hydrogenase. Three of these are Ni proteins and one contains iron as the only transition metal. These proteins catalyze the reversible oxidation of dihydrogen gas (Eq. 7). A fifth class of hydrogenase lacks metals and catalyzes Eq. 8, but not Eq. 7. The Ni enzymes are classed into the NiFe, the NiFeSe, and the hydrogen sensors. All of these enzymes are bidirectional, although different enzymes exhibit a catalytic bias toward making or oxidizing H2. 

FIGURE 1. Cartoon of the two active sites of the bifunctional CO dehydrogenase/acetyl-CoA 

However, recent studies have shown that especially with [NiFe] hydrogenases some kind of O2 tolerance can be observed, where in most cases O2 behaves as a reversible inhibitor rather than an agent that effectively causes irreversible inactivation [34]. 

Although the results shown in this study provide only a proof-of-principle demonstration of such a system, it may be foreseen that, given the availability of several characterized hydrogenases [33], new hydrogenase-diaphorase cofactor regeneration particles will be investigated in the future. 

For example, a ferredoxin hydrogenase (EC 1.12.7.2) has been isolated recently from the hyperthermophile Pyrococcus fUriosus [38]. The performance of this biocatalyst, which showed a remarkable stability under operative conditions, has been investigated for the NADPH regeneration in the reduction of prochiral ketones catalyzed by the thermophilic NADPH-dependent ADH from Thermoanaerohium sp. Total turnover numbers (TTNs mole product/mole consumed cofactor NADP ) of 100 and 160 could be estimated in the reduction of acetophenone and (2S)-hydroxy-l-phenyl-propanone, respectively. As a side note, it should be mentioned that, although the activity of the P. furiosus hydrogenase increased exponentially with temperature up to its maximum above 80 °C, the reactions had to be performed at much lower temperature (40 °C) because of the thermal instability of NADPH. 

In contrast to urease the nickel in other bacterial enzymes appears to have a redox function and to take up oxidation states Ni(I) and/or Ni(III). Fortunately these states have recently become better understood in inorganic systems (see the preceding review in this volume by 

Types of iron-sulfur clusters and other groups, such as flavin or selenium, that are present. 

Sensitivity to deactivation by oxygen and, in some cases, slow, reductive reactivation. 

Different ratio of products in hydrogen isotope-exchange assays. 

Among the diversity of Ni hydrogenases, there is a common pattern of protein composition, to which most conform, which consists of two protein subunits of relative molecular mass approximately 60,000 and 30,000 Da (14). There is some evidence (38) that the nickel is situated in the 60,000-Da subunit. More complex hydrogenases, such as the soluble hydrogenase of Nocardia opaca (Table I), contain other subunits which are concerned with the reduction of specific electron acceptors. 

Cytochrome c3 is characteristic of the sulfate-reducing bacteria. The cytochrome has four heme C molecules in the molecule (13 kDa) (Yagi and Maruyama, 1971). Both the 5th and 6th axial ligands of the four heme C molecules in the cytochrome are histidine residues at pH 6.0(Higuchi et al., 1981). The midpoint redox potentials at pH 7.0 of the four heme molecules vary with the heme ranging from 

It was shown in 1968 (Yagi et al., 1968) that cytochrome c3 acts as the electron donor for hydrogenase. However, it was not until 1994 that the cytochrome was found to function perfectly as the electron donor for sulfite reductase. Steuber et al. (1994) has demonstrated that sulfite reductase solubilized with the aid of a detergent from D. desulfuricans catalyzes the reduction of sulfite to hydrogen sulfide without any intermediates using reduced cytochrome c3 as the electron donor. 

In the sulfate-reducing bacteria there is another cytochrome which resembles cytochrome c3 in the spectral properties and redox potential but differs from this cytochrome in molecular mass this is cytochrome c3 (26 kDa) which has eight heme C molecules in the molecule. The cytochrome molecule is composed of two polypeptides of 13 kDa (Loufti et al., 1989). On the basis of the amino acid sequence, however, the 13 kDa polypeptide differs from cytochrome c3 (Guer-lesquin et al., 1982 LeGall and Peck, 1987 Loufti et al., 1989). Desulfovibrio gigas cytochrome c3 (26 kDa) molecule is composed of two 13 kDa molecules bound to each other by an S-S bond (Bruschi et al., 1996). It is claimed that cytochrome c3 (26 kDa) is very effective as the electron donor for thiosulfate reductase (Hat-chikian et al., 1972). 

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Oxidoreduciases. Enzymes catalysing redox reactions. The substrate which is oxidized is regarded as the hydrogen donor. This group includes the trivially named enzymes, dehydrogenases, oxidases, reductases, peroxidases, hydrogenases and hydroxylases. 

The electrons undergo the equivalent of a partial oxidation process ia a dark reaction to a positive potential of +0.4 V, and Photosystem I then raises the potential of the electrons to as high as —0.7 V. Under normal photosynthesis conditions, these electrons reduce tryphosphopyridine-nucleotide (TPN) to TPNH, which reduces carbon dioxide to organic plant material. In the biophotolysis of water, these electrons are diverted from carbon dioxide to a microbial hydrogenase for reduction of protons to hydrogen ... 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

It can be seen from equation 2.14 that the ratio of iron corroded to iron in the form of sulphide should be 4 1, but values from 0.9 to 48 are commonly obtained experimentally. Subsequently it was shown by Booth and his co-workers that the ratios of the corrosion products were dependent on the particular strain of Desulphovibrio and on their rates of growth. Later the activity of the enzyme hydrogenase which bring about the reaction ... 

This enzyme is of wide occurrence in bacteria where it is concerned with the reduction of nitrate and CO2 as well as sulphur. Methods for its estimation depend on measuring some activity of hydrogenase by (a) dye reduction (benzyl viologen or methylene blue), (b) isotopic exchange and (c) evolution of molecular hydrogen. Interpretation of quantitative results is difficult due to the complex relationship between the enzyme cell structure and the particular method selected. ... 

Hydrogenase and its application for photoinduced hydrogen evolution. I. Okura, Coord. Chem. Rev., 1985,68,53 (141). 

Frenking G, Cremer D (1990) The Chemistry of the Nobles Gas Elements Helium, Neon, and Argon - Experimental Facts and Theoretical Predictions. 73 17-96 Frey M (1998) Nickel-Iron Hydrogenases Structural and Functional Properties. 90 97-126 Fricke B (1975) Superheavy Elements. 21 89-144... 

NiFe-hydrogenase Bacteria H2 2H + 2e- [FesS4] 2[Fe4S4p- NiFe center -70 59... 

Fig. 1. Proposed electron transport pathway in D. gigas NiFe-hydrogenase. Selected distances are given in angstroms. Modified with permission from Ref. (157).

The spatial arrangement of the Fe-S clusters in D. gigas NiFe-hydrogenase (see Fig. 1) suggests an active role for the [Fe3S4] ° cluster in mediating electron transfer from the NiFe active site to the... 

The multifrequency EPR and Mdssbauer properties of the [FesSJ in C. vinosum NiFe-hydrogenase are particularly interesting since they provide evidence of magnetic interactions with nearby paramagnetic species (151, 154, 155). The magnetically isolated form exhibits a well-resolved, almost axial EPR signal, g = 2.018, 2.016, 2.002, indicative of minimal conformational heterogeneity. However, a com-... 

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