Printer Friendly

Studies on hydrogenase.


Hydrogenase (1,2) was first described by Stephenson and Stickland as an enzyme which catalyzed activation of [H.sub.2] to reduce electron acceptors such as [O.sub.2], nitrate, fumarate, sulfate, or an artificial electron acceptor, methylene blue. Some hydrogenases catalyze the reverse reaction to produce [H.sub.2] from electron donors such as methylviologen cation radical (reduced methylviologen), and are called reversible hydrogenases, whereas the enzymes unable to catalyze the [H.sub.2]-production are called uptake hydrogenases. Hydrogenases also catalyze isotopic exchange between dihydrogen and water, and conversion between para-[H.sub.2] and ortho-[H.sub.2]. (3-6) Hydrogenases are widely distributed in Bacteria and Archaea. (7) In Eucarya, hydrogenases are found in unicellular algae (8) and hydrogenosomes of unicellular Protozoa. (9) Recent discovery (10) of hydrogenosome like organelles in three species of animal phylum, Loricifera, living in sediments of the deep anoxic hypersaline L'Atalante basin, makes us expect for the presence of hydrogenase in animals.

Classification of hydrogenases

Classification based on the electron carrier specificity. During a few decades after the discovery (1,2) of hydrogenase in 1931, the enzymatic activity had been demonstrated with artificial electron carriers such as methylviologen, benzylviologen or methylene blue, or by isotope exchange reactions, and the natural electron carriers had been out of concern. Mortenson et al. (11) announced the discovery of ferredoxin (Fd), a small iron-sulfur (FeS) protein of low redox potential, from Clostridium pasteurianum, and demonstrated it to be a physiological electron carrier for clostridial hydrogenase (reaction 1). This enzyme is now registered as ferredoxin hydrogenase [Fd hydrogenase, EC] by IUBMB (International Union of Biochemistry and Molecular Biology,

[H.sub.2] + Fd [??] 2[H.sup.+] + reduced Fd [1]

Purified hydrogenase from hydrogen bacterium (12,13) was reported to catalyze reduction of NADD in the absence of any additional cofactor, but the possibility of this hydrogenase to act on Fd had not been tested. This enzyme was named hydrogen dehydrogenase [EC].

Hydrogenase from sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki (DvM), was shown to act on cytochrome [c.sub.3] (cyt-[c.sub.3]) reversibly (reaction 2) but not on Fd, whereas clostridial hydrogenase was shown not to act on cyt-[c.sub.3]. (14,15) Hence the name cyt-[c.sub.3] hydrogenase [EC] was given to DvM hydrogenase. This established that hydrogenases from different origins may have different specificity to their electron carriers. Cyt-[c.sub.3] is a basic tetrahemoprotein of 14kDa, (16)) and its four hemes are reduced successively (17)) according to the redox potentials (-280, -321, -325 and -356mV). (18,19))

2[H.sub.2] + ferricyt-[c.sub.3] [([Fe.sup.3+]).sub.4] [??] 4[H.sup.+] + ferrocyt-[c.sub.3] [([Fe.sup.2+]).sub.4] [2]

Kinetic parameters for this reaction are: [K.sub.m]([H.sub.2], pH 6.0) (20)) f 16.5 [micro]mol [L.sup.-1] (2.2 kPa [H.sub.2]), [K.sub.m](ferricyt-[c.sub.3], pH 7.0) (21)) f 2.6 [micro]mol [L.sup.-1], [K.sub.m](ferrocyt-[c.sub.3], pH 6.0) (20)) f 17 [micro]mol [L.sup.-1], [] (for [H.sub.2] uptake, pH 7.0) (21)) f 49 [s.sup.-1] and [] (for [H.sub.2] evolution with saturating cyt-[c.sub.3] concentration, pH 6.0) (22)) = 336 [s.sup.-1]. Hydrogenases from other sulfate-reducing bacteria such as D. vulgaris Hildenborough (DvH), (23)) D. gigas (Dg), (24)) etc., act on their respective cyt-[c.sub.3]s. Now, 10 kinds of hydrogenases with different electron carrier specificity are registered by IUBMB as shown in Table 1.

Some non-hydrogenase enzymes catalyze [H.sub.2] uptake or evolution. Nitrogenases [EC and] produce [H.sub.2] as a by-product in the normal reaction to reduce [N.sub.2] to ammonia, and as a main product in the absence of [N.sub.2] at the expense of ATP, (27)) carbon monoxide dehydrogenase/acetyl-CoA synthase [EC d] and pyruvate: Fd oxidoreductase [EC] reduce [H.sup.+] to [H.sub.2] and, at much lower rate, oxidize [H.sub.2] to [H.sup.+], (28)) and bacterial alkaline phosphatase [EC] catalyzes hydrolytic cleavage of the H-P bond of inorganic phosphonate (HP[O.sub.3.sup.2-]) to produce [H.sub.2] and phosphate (HP[O.sub.4.sup.2-]). (29)

Classification based on the structure of the active site. All hydrogenases except [EC]-enzymes can be classified into two families based on the structure of the active sites, i.e., [NiFe]-hydrogenases (active site: binuclear Ni-Fe center) and [FeFe]-hydrogenases (active site: binuclear Fe-Fe center). There is no correlation between the type of the active site and the electron carrier specificity. For example, cyt-[c.sub.3] hydrogenases from DvM and Dg are of [NiFe]-type, (24),30)) whereas those from DvH and Desulfovibrio desulfuricans are of [FeFe]-type, (31,32) and Fd hydrogenase from C. pasteurianum is of [FeFe]-type, (33)) whereas that from Methanosarcina is of [NiFe]-type. (34) Some [NiFe]-enzymes have a selenocysteinyl (Sec) residue instead of one of the Cys residues supporting the Ni-Fe center, (35) and are called [NiFeSe]-hydrogenases.

The third family of hydrogenases, [Fe]-hydrogenases (active site: a mononuclear Fe center), consists of only [EC]-enzymes from methanogenic Archaea (Attention: in literature before ca. 2002 when these enzymes had been believed to be metal-free enzymes, (36,37) [Fe]-hydrogenases meant today's [FeFe]-hydrogenases).

H-D exchange and para-[H.sub.2]-ortho-[H.sub.2] conversion

The H-D exchange and the conversion between para-[H.sub.2] and ortho-[H.sub.2] are regarded as fundamental reactions of hydrogenases, because these reactions can be observed in the absence of any electron carrier. Rittenberg and Krasna (3,4) used these reactions to elucidate the mechanism of action of hydrogenase of Proteus vulgaris, and proposed that the cleavage of [H.sub.2] by the enzyme (E) was heterolytic to form two unequal parts (reaction 3).

[H.sub.2] + E [??] [H.sup.+] + E:[H.sup.-] [3]

Here, [H.sup.+] is readily exchangeable with the medium [D.sup.+] in [D.sub.2]O, but the enzyme-bound hydride (E : [H.sup.-])is not. This conclusion was based on the observation that the ratio of [D.sub.2] and HD initially produced from [H.sub.2] over [D.sub.2]O was determined to be nearly zero ([v.sub.DD]/ [v.sub.HD] f 0 from [H.sub.2]/[D.sub.2]O), (4)) and that the conversion of para-[H.sub.2] to ortho-[H.sub.2] did not occur over [D.sub.2]O whereas the isotope exchange occurred ([v.sub.p-o]/[v.sub.ex] f 0 from p[H.sub.2]/[D.sub.2]O, where [v.sub.ex] f [v.sub.HD] d [v.sub.DD]).3) If both of the cleaved H species are partially exchangeable (i.e., partially unexchangeable) with the medium DD, either [v.sub.DD]/[v.sub.HD] or [v.sub.p-o]/[v.sub.ex] must have not been zero. Tamiya and Miller (5)) reexamined these reactions and found that [v.sub.DD]/[v.sub.HD] from [H.sub.2]/[D.sub.2]O was dependent on the enzyme concentration, and was 0.19 at the enzyme concentration extrapolated to 0 for P. vulgaris enzyme. The [v.sub.DD]/[v.sub.HD] from [H.sub.2]/[D.sub.2]O was dependent on pD of the reaction medium, as well as on the bacterial source of the enzyme (e.g., 0.45 for C. pasteurianum enzyme at pD 7.0). The [v.sub.p-o]/[v.sub.ex] from p[H.sub.2]/[D.sub.2]O was determined to be below the limit of detection (<0.1) for enzymes from Proteus and Clostridium.

The initial [v.sub.DD]/[v.sub.HD] and [v.sub.p-o]/[v.sub.ex] ratios from p[H.sub.2]/[D.sub.2]O, as well as the [v.sub.HH]/[v.sub.HD] from [D.sub.2]/[H.sub.2]O were reexamined by Yagi et al. (6)) when the DvM hydrogenase was purified (22)) (in retrospect, the purified enzyme contained about 70% of the active form, the remainder being inactive forms of the enzyme, vide infra), and simple and accurate assay of para-[H.sub.2] and ortho-[H.sub.2] became available. The exchange and the conversion reactions by the enzyme can be observed in the absence of added cyt-[c.sub.3], but much higher reaction rates were observed reproducibly in its presence (ferrocyt-[c.sub.3] was later (21)) found to influence the electronic structure of hydrogenase), hence the reactions were carried out in its presence. The [v.sub.DD]/ [v.sub.HD] and [v.sub.HH]/[v.sub.HD] ratios were 0.20 and 0.40, respectively, at the enzyme concentration extrapolated to 0, but were 1.5 and 3.6, respectively, when the enzyme concentration was extremely high, because the dihydrogen species liberated from the enzyme had additional chances to be caught by the enzyme for additional exchange reactions (cage effect) (5) before release to the gas phase. The [v.sub.p-o]/[v.sub.ex] ratio was higher at lower enzyme concentration, and were as high as 0.7 at the enzyme concentration extrapolated to 0. These experimental results were successfully explained by the mechanism schematically represented in Fig. 1. The kinetic parameters which are compatible with these experimental data were calculated, and given in the figure. This scheme conforms to the heterolytic cleavage of the enzyme-bound [H.sub.2], i.e., one of the enzyme-bound [H.sub.2] has the rate constant ([k.sub.a]) for the isotope exchange reaction 10 times as that ([k.sub.b]) of the other, but the mode of heterolysis is not as simple as what was initially suggested as in reaction 3. (3,4) Any structural model proposed for the enzyme-bound [H.sub.2] must conform to the scheme shown in Fig. 1.

The catalytic mechanism of hydrogenase proposed in Fig. 1 suggests that water does not participate in para-[H.sub.2]--ortho-[H.sub.2] conversion. In fact, dry hydrogenase was proved to catalyze the conversion reaction at a reaction rate (k = 1.3 [s.sup.-1]) about 1/340 of that in aqueous reaction in the absence of cyt-[c.sub.3]. (38)) The isotopic exchange reaction between [H.sub.2] and [D.sub.2] to produce HD was also observed with dried hydrogenase--cyt-[c.sub.3] mixture with a reaction rate 20% that of the conversion reaction. (39) This indicates that the covalent bond of [H.sub.2] is split on the enzyme molecule and the bound H ([H.sub.a] or [H.sub.b], or both) is exchangeable with hydrons ([H.sup.+] or [D.sup.+]) on the protein molecule. When a mixture of hydrogenase and excess cyt-[c.sub.3] (400 times in molar base, i.e., 63 times by mass) are lyophilized and placed under the atmosphere of [H.sub.2] at 102 kPa, cyt-[c.sub.3] was reduced nearly completely, and partially reoxidized on evacuation of [H.sub.2]. (39)) These results indicate that hydrogenase is active in anhydrous state, that the enzyme has exchangeable hydrons, and that cyt-[c.sub.3] molecules in the reduced state can transfer electrons and hydrons to other cyt-[c.sub.3] molecules, i.e., ferrocyt-[c.sub.3] is proteinaceous conductor. In fact, semiconductive nature of anhydrous ferrocyt-[c.sub.3] was proven by direct measurements of conductivity. (40,41)

Development of continuous mass-spectroscopic monitoring system (42) of isotopic dihydrogens greatly simplified the exchange studies. Table 2 summarizes the results of isotope exchange and para-[H.sub.2]--ortho-[H.sub.2] conversion reactions reported in literature. The [v.sub.DD]/ [v.sub.HD] (from [H.sub.2]/[D.sub.2]O) or [v.sub.HH]/[v.sub.HD] (from [D.sub.2]/[H.sub.2]O) varies significantly among hydrogenases from different origins. Extremely higher [v.sub.DD]/ [v.sub.HD] or [v.sub.HH]/ [v.sub.HD] ratios observed with membrane particles, organelles or cells must have been due to the cage effect,5) because enzymes are expected to be locally concentrated, or free diffusion of dihydrogen species are limited there. For the purified [NiFe]- and [FeFe]-enzymes, the [v.sub.DD]/ [v.sub.HD] ratios are 0.2-0.4 with few exceptions, and the [v.sub.HH]/ [v.sub.HD] ratios are 0.3-0.6, with a notable exception for [NiFeSe]-enzyme. These data may be considered proof for the heterolytic cleavage of dihydrogen, but strictly speaking, [k.sub.a]/[k.sub.b] ratio can be calculated only when [v.sub.DD]/[v.sub.HD] and [v.sub.p-o]/[v.sub.ex] ratios (from p[H.sub.2]/ [D.sub.2]O) were measured simultaneously. For example, [v.sub.DD]/[v.sub.HD] ratio could be 0.2 for [H.sub.2] homolysis ([k.sub.a] = [k.sub.b] = 0.18[k.sub.-1] in Fig. 1) if [v.sub.p-o]/[v.sub.ex] were determined to be 2.8.

Higher [v.sub.DD]/ [v.sub.HD] or [v.sub.HH]/[v.sub.HD] ratios were observed for Ni-rubredoxin (rubredoxin with its tetrahedrally complexed [Fe.sup.2+/3+] ion substituted with [Ni.sup.2+] ion) (51)) and a binuclear NiRu complex ([Ni.sup.II][([mu]-SR).sub.2]([mu]-H)[Ru.sup.II]). (52) The mode of [H.sub.2] splitting by Ni-rubredoxin, which had been prepared before the structure of the Ni-Fe center was elucidated, could either be homolytic (e.g., [k.sub.a] = [k.sub.b] = 1.3[k.sub.-1] in Fig. 1) or heterolytic (e.g., ka f 3kb = 3[k.sub.-1]) depending on the [v.sub.p-o]/[v.sub.ex] ratio which had not been determined. This complex also catalyzed production of [H.sub.2] from methylviologen cation radical. As the catalytic activities of Ni-rubredoxins depended on the sources of rubredoxin, (51)) the protein part must have been important for the catalytic function. The mode of [H.sub.2] splitting by Ni-Ru complex, (52)) which had been designed to mimic the Ni-Fe center of [NiFe]-hydrogenase, could also be either homolytic (e.g., [k.sub.a] = [k.sub.b] = 3.1[k.sub.-1]) or heterolytic (e.g., [k.sub.a] = 2[k.sub.b] = 5[k.sub.-1]). This complex catalyzed reduction of ketone to alcohol with [H.sub.2].

Amino acid sequence and subunit composition of hydrogenases

The amino acid sequence of DvH [FeFe]-hydrogenase was first reported by Voordouw. (31) (Database accession: P07598 and P07603 for the heterodimer). The sequences of Dg [NiFe]-hydrogenase (Fig. 2), Desulfomicrobium baculatum [NiFeSe]-enzyme (Fig. 2), C. pasteurianum [FeFe]-enzyme (P29166) and Methanocaldococcus jannaschii [Fe]-enzyme (Q58194) were then reported. The sequences of [NiFe]-family, [FeFe]-family and [Fe]-family enzymes are phylogenetically unrelated. It is intriguing to note that, whereas majority of hydrogenases expressed in Bacteria and Archaea are of [NiFe]-type, (7) only [FeFe]-enzymes are expressed in Eucarya.

Every [NiFe]-hydrogenase is composed of a catalytically active core heterodimer, with or without additional subunit(s). Cyt-[c.sub.3] hydrogenases from sulfate reducing bacteria (Desulfovibrio, Desulfomicrobium, etc.) have no additional subunit, (35,53,54) and are the simplest among the [NiFe]-family. Quinone-reducing hydrogenase (7,55) and methanophenazine hydrogenase (7,56,57) have membrane-bound dihemic cytochrome b as the third subunit to interact with the electron carrier, menaquinone and methanophenazine, respectively. [F.sub.420]-reducing hydrogenase from methanogenic archaeon is an oligomer of trimers consisted of the core heterodimer and another subunit. (58) [NAD.sup.+]-reducing hydrogenase from hydrogen bacterium, Ralstonia eutropha H16 has four more subunits, two of which constitute diaphorase to mediate electron transfer between the core hetero dimer and [NAD.sup.+]/NADH. (59) The sequence alignments of the core heterodimers of some [NiFe]-hydrogenases (Fig. 2) indicate that they are homologous regardless whether the enzymes are of archaeal (No. 6) or bacterial origins, or they are cytoplasmic (No. 7) or periplasmic.

[FeFe]-hydrogenase may be a monomer (Fd hydrogenase from C. pasteurianum, (33,63) Mega-sphaera elsdenii, (64) etc.), a heterodimer (e.g., DvH cyt-[c.sub.3] hydrogenase (31)), a heterotrimer (e.g., bifurcating hydrogenase (65)), or a heterotetramer (e.g., D. fructosovorans [NADP.sup.+]-reducing hydrogenase (66)). [Fe]-hydrogenase is a homodimer composed of only 1 gene product. (67,68)

Three-dimensional structure of hydrogenases

The X-ray structure of Dg [NiFe]-hydrogenase was elucidated by French group, (69,70) and then that of DvM [NiFe]-enzyme (Fig. 3a), by the authors. (71) The structures of [FeFe]-enzymes from C. pasteurianum (72) and D. desulfuricans, (32) [NiFeSe]-enzyme from D. baculatum, (73) and [Fe]-enzyme from M. jannaschii (68) were successively determined.

Standard [NiFe]-hydrogenase. Hydrogenases from DvM and Dg are very similar in the main-chain folding, reflecting their sequence homology (67% identical, Fig. 2). The Ni-Fe center is held by 4 Cys residues of the large subunit, and is buried deep in the center of the protein. (69-71) The Fe atom of the Ni-Fe center is coordinated by 3 diatomic ligands (Fig. 3b). (74) An additional metal center, [Mg.sup.2+]-center, also held by the large subunit was revealed by 1.8 [Angstrom] resolution crystallography. (71) Three FeS clusters, i.e., proximal and distal [4Fe-4S] clusters and mesial [3Fe-4S] cluster from the Ni-Fe center are held by the small subunit, and align linearly. The binding site for cyt-[c.sub.3] was suggested to locate near the distal [4Fe-4S] cluster (Fig. 3a). (21) [H.sub.2] is supposed to access the Ni-Fe center through hydrophobic channels connecting the Ni-Fe center to the protein surface. (75-77)

The structures of Ni-Fe centers from Dg and DvM enzymes are similar, but subtly differ from each other. The bridging ligand (purple sphere in Fig. 3b) between Ni and Fe was assigned as oxygen species (OH or [O.sub.2.sup.-]) for Dg enzyme, (70) but as sulfur species ([S.sup.-] or SH) for DvM enzyme. (71) This might reflect the fact that DvM enzyme had been purified under strict anaerobiosis and was active as isolated, whereas Dg enzyme was prepared under aerobiosis as an inactive unready state, which could be activated only after prolonged incubation under [H.sub.2]. (24) It was later established that the bridging ligand of the unready and ready forms were hydroperoxide and hydroxo species for D. fructosovorans enzyme, (78) and disappeared upon reductive activation. (79) When DvM enzyme was incubated under [H.sub.2] in the presence of cyt-[c.sub.3], [H.sub.2]S was liberated (80) and the enzyme lost the bridging sulfur species at the Ni-Fe center. (81) DvM enzyme, on oxidation, was shown to have [OH.sup.-] as the bridging ligand by EPR and electron-nuclear double resonance spectroscopy applied to single crystals of the enzyme, (82) and the bridging ligands of the Ni-A and Ni-B forms of DvM enzyme were suggested to be peroxo (or hydroperoxo) and oxo species, respectively. (83)

Atomic species of three diatomic ligands to Fe are enigmatic. These were assigned as 1 CO and 2 [CN.sup.-] for Dg enzyme, (74) as well as for similar [NiFe]-enzyme from Chromatium vinosum, (84) whereas the electron density of one of three ligands (L1 of Fig. 3b) of DvM enzyme was too big to be modeled as [CN.sup.-] or CO, and had to be assigned as SO. (71) The presence of a rather bizarre SO ligand had precedents in synthetic coordination compounds, (85) and SO (m/z = 48) began to be released from the native DvM enzyme by mass spectrometry at about 400 K, (86) far below the decomposition temperature of covalent bonds in proteins and FeS clusters. However, later enzyme lots prepared from different batches of the bacterium had the diatomic ligands similar in size to those in Dg enzyme. (87) Possibility of erroneous assignment as SO in the earlier study (71) is improbable unless other source of SO is specified, because SO was detected by the technique (86) different from X-ray crystallography. Subtle difference in culturing bacterium might have affected the diatomic ligand composition, but the way how the culturing conditions affect the ligand composition has not yet been figured out. Some residual electron densities (not fully occupied monatomic species, O or S) were observed near the S atoms of some Cys residues (Cys84 and Cys546 for DvM enzyme). (75,83,87)

[O.sub.2]-tolerant [NiFe]-hydrogenase. Membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus is extremely thermophilic and functions in the presence of [O.sub.2], (88)) the properties unique among hydrogenases. The Ni-Fe center of this enzyme is similar to that of the standard [NiFe]-enzyme from DvM or Dg (Fig. 3b), but the proximal FeS cluster is not of [4Fe-4S]-type, but of [4Fe-3S]-type (Fig. 4). (60) The Gly residue in the conserved CTG*CS (G asterisked to be highlighted) segment at the binding site to the proximal [4Fe-4S] cluster in the standard [NiFe]-enzyme is substituted by Cys65, and the 2nd Gly residue in the conserved GG*VQAA segment also at the binding site to the same [4Fe-4S] cluster in the standard enzyme is substituted by Cys166 residue (CTC*CS and GC*VQAA segments of the small subunit in Fig. 2). Instead of the missing [S.sup.2-] ion which would have coordinated to Fe1, Fe2 and Fe4 of the standard enzyme, Cys65-thiolate coordinates to Fe1 and Fe2, and Cys166-thiolate coordinates to Fe4, to make the proximal [4Fe-3S] cluster in the reduced form of H. marinus enzyme (Fig. 4a). In the standard [4Fe-4S] clusters, each Fe ion in [4Fe-4S] clusters is held by a single Cys-thiolate (Fig. 4c), but in the [4Fe-3S] cluster of H. marinus enzyme, each of three Fe ions (Fe1, Fe2 and Fe4) is doubly coordinated by 2 Cys-thiolates, therefore the [4Fe-3S] cluster is more stably embraced by the protein than the standard [4Fe-4S] clusters. On oxidation of the enzyme, the vertical Fe2-S3 bond of the [4Fe-3S] cluster in Fig. 4a is cleaved, and Fe2 becomes coordinated by amide [N.sup.-] of Cys66 backbone, resulting in significant conformational change of the cluster (Fig. 4b), without being thrown out from the protein. The cleavage of the Fe2-S3 bond makes the highly conjugated system of the [4Fe-3S] cluster to loosely conjugated 2 subparts, to enable the [4Fe-3S] cluster to conduct two-electron redox change to reduce the oxygen species introduced to the Ni-Fe center during the catalytic cycle in the presence of [H.sub.2] and [O.sub.2]. Another membrane-bound [O.sub.2]-tolerant [NiFe]-hydrogenase from R. eutropha (89)) (P31892, not the soluble enzyme 7 in Fig. 2) has a similar [4Fe-3S] cluster held by the same CTC*CS and GC*VQAA sequences (90)) as those of H. marinus hydrogenase, and the mutation of both [Cys.sup.*] residues to Gly converted the enzyme to a standard [O.sub.2]-sensitive hydrogenase. (89))

[NiFeSe]-hydrogenase. The structure of [NiFeSe]-hydrogenase from D. baculatum in the reduced form, (73)) is similar to the standard [NiFe]enzymes from DvM and Dg, except that one of Cys residues coordinated to the Ni atom is replaced by Sec (U493 in Fig. 2), the monatomic center has Fe instead of Mg in the large subunit, and a [4Fe-4S] cluster instead of the mesial [3Fe-4S] cluster in the small subunit. The three diatomic ligands to Fe are 1 CO and 2 [CN.sup.-] as in the case of the standard enzymes, but the bridging site between Ni and Fe is vacant as isolated. Intriguingly, at position 6.7 [Angstrom] from the Ni-Fe center, [H.sub.2]S is retained, which might have been liberated from the Ni-Fe center, but these authors (73) argued against the ex-presence of sulfur species at the Ni-Fe bridging site. The [NiFeSe]-hydrogenase as isolated from DvH also has a vacant bridging site between Ni and Fe. (76)) [NiFeSe]-enzymes are known to be [O.sub.2]-tolerant, but the mechanism of [O.sub.2]-tolerance was suggested to be due to prevention of [O.sub.2] accession through [H.sub.2] channels by Sec-selenol and monatomic extra atom near the bridging Cys residue to Ni and Fe. (73,76) In addition, its proximal [4Fe-4S] cluster has features different from those of the standard [NiFe]-enzymes. (76)


[FeFe]-hydrogenase. Heterodimeric [FeFe]-hydrogenase from D. desulfuricans (32,91) differs from the [NiFe]-enzyme in protein folding. The active site called H-cluster is composed of a binuclear Fe-Fe center and a [4Fe-4S] cluster, both of which are held by a Cys thiolate of the large subunit, and is buried deep in the center of the protein. Instead of 2 Cys residues bridging Ni and Fe atoms of the Ni-Fe center in the [NiFe]-enzyme, two dithiolate groups of 2-(aza or oxa)propane-1,3-dithiol are bridging two Fe atoms of the Fe-Fe center (Fig. 5a). Two other [4Fe-4S] clusters are also held by the large subunit. In spite of these differences from the [NiFe]-enzyme, the Fe atoms at the Fe-Fe center coordinate diatomic CO and [CN.sup.-] ligands as in the Ni-Fe center of the [NiFe]-enzyme. The monomeric [FeFe]-enzyme from C. pasteurianum has a similar H-cluster as that of D. desulfuricans enzyme, but differs in having extra [4Fe-4S] and [2Fe-2S] clusters. (72)

[Fe]-hydrogenase. The active site of [Fe]-hydrogenase locates on the protein surface, has ligands of 6-carboxymethyl-4-(5/-guanylyloxy)-3,5dimethylpyridin-2-ol and 2 CO, and is supported by Cys-thiolate of the protein (Fig. 5b). Unlike the hydrogenases of the other families, both [H.sub.2] and the electron mediator, 5,10-(methenyl/methylene)tetrahydromethanopterin bind at the active site to react directly without participation of FeS clusters. (68))

Catalytic mechanism of [NiFe]-hydrogenase

Activation of the enzyme. Dg [NiFe]-hydrogenase as purified aerobically is in the inactive unready state, which is activated only after prolonged incubation under [H.sub.2]. (24) The activated enzyme can be oxidized anaerobically to the ready state, which is inactive but readily activated on reduction. The unready, ready and active states were correlated to the EPR signals dubbed Ni-A (g = 2.31, 2.26, 2.02), Ni-B (g = 2.33, 2.16, 2.02) and Ni-C (g = 2.19, 2.14, 2.02), respectively. (92) The interfering signal of the mesial [3Fe-4S] cluster can be quenched by measuring EPR at temperature above 50 K. The Fe atom is diamagnetic at any redox state of the enzyme. (93) FTIR spectroscopy, in combination with EPR and electrochemistry distinguished different states of the enzyme, including the EPR silent states. (74) The activation/ inactivation pathways of the enzyme in various redox states are illustrated in Fig. 6 (upper 2 rows), where direct oxygenation of Ni-B to Ni-A form has not been observed with Dg enzyme. (70) Activation of Ni-[B.sub.1946] (see the legend to Fig. 6 for the subscript) to Ni-[SI.sub.1914] form may not proceed in one step as indicated, but Ni-[B.sub.1946] may be reduced (+ [e.sup.-]) followed by the removal of the bridging ligand (-O[H.sup.-]). (77)

DvM [NiFe]-hydrogenase is active or readily activated as isolated. (53,71) Some preparations had only an EPR signal at g = 2.017 of [3Fe-4S] cluster, whereas others had additional signals (g = 2.32, 2.23, 2.16, 2.01) of combined Ni-A and Ni-B forms. (94) It was later recognized that the accession of air to the enzyme during purification was not carefully controlled at that time, so that some preparations contained detectable amounts of Ni-A (g = 2.32, 2.24, 2.01) and Ni-B (g = 2.33, 2.16, 2.01) forms, (30,83,95) but others contained hardly detectable amounts of Ni-A and Ni-B forms. The enzyme purified under aerobiosis contained 70% Ni-B form, and behaved as an active enzyme in the assay system, in which the enzyme was reduced chemically with dithionite (14,15,22) or electrochemically (96) to start the reaction. The redox titration of the enzyme having EPR Ni-signals with sodium dithionite led to successive disappearance of Ni-B and Ni-A signals at midpoint potentials of -230 and -310 mV, respectively. Ni-C signal (g = 2.196, 2.144) appeared at potential -370 mV, reached a maximum at about -400 mV, and disappeared at -430 mV. (30) Ni-B form (having oxo or hydroxo species as the bridging ligand between Ni and Fe) can be converted to Ni-A form (having peroxo or hydroperoxo species as the bridging ligand) by treatment with [Na.sub.2]S to form Ni-B' (g = 2.29, 2.14, 2.00), followed by the addition of [O.sub.2]. (83) Some residual electron densities observed near the S atoms of Ni-Fe-supporting Cys residues, (83,87) might be related to some behaviors of the enzyme (e.g., evolution of [H.sub.2]S on activation, activation/inactivation of various states of the enzyme, etc.), but they are disregarded in the following discussion, because we do not know the real structure at work in vivo.

(71) (77) (81) (30) (74) (77)

It is noteworthy that in spite of differences in the bridging ligand between Ni and Fe, both of DvM and Dg enzymes, on activation, behave similarly in redox processes. (30)

Catalytic cycle. Based on the kinetic, structural and spectroscopic studies described in the preceding sections, the catalytic cycle of [NiFe]-hydrogenase was proposed to explain [H.sub.2]-uptake, [H.sub.2]-production and isotopic exchange reactions, as shown in the lower part of Fig. 6. (97) Only the 3 structures present in the lower pH range (i.e., protonated forms shown in the shaded boxes in the inner circuit of the catalytic cycle) were proved to be catalytically active. (74) We think, however, that both protonated and deprotonated Ni-[SI.sup.(-)] forms have catalytic activity and the inner and outer circuits of the catalytic cycle are operating, because hydrogenase is active in a wide pH range, and the pH optimum for [H.sub.2] evolution (p[H.sub.opt][up arrow]) is lower than the p[K.sub.a] between Ni-[SI.sup.(-).sub.1934] and Ni-[SI.sup.(-).sub.1914], that of [H.sub.2] uptake (p[H.sub.opt][down arrow]) is higher than that p[K.sub.a], and that of the isotopic exchange is between p[H.sub.opt][up arrow] and p[H.sub.opt][down arrow]. (15),22),45)-49))

[H.sub.2] diffused from the protein surface through hydrophobic tunnels (75-77) is suggested to bind at the 6th ligand site of Ni of Ni-SI forms of the enzyme, because gaseous CO, the competitive inhibitor, (15) was found to bind at this site, (87) but see the legend to Fig. 6 for other possibilities. Inactivity of Ni-A form can be explained by the fact that the outer atom of the diatomic bridge of this form occupies the same position as that of CO in the CO-bound enzyme to prevent [H.sub.2] from binding to Ni. Heterolytic cleavage of the bound [H.sub.2] produces Ni-R form, in which the hydride is believed to bind at the Ni-Fe bridging site, (77,98-100) and [H.sup.+] on the S atom of [Cys.sup.*] ([Cys.sup.*] is Cys546 for DvM enzyme). (93) In the [H.sub.2]-uptake reaction to reduce ferricyt-[c.sub.3], the outer circuit starting from Ni[SI.sub.1914] form operating clockwise to produce (Ni-R) form (deprotonated form of Ni[R.sub.1940], not confirmed by spectroscopy) must be predominant, because p[H.sub.opt][down arrow] lies above the p[K.sub.a]. The resulting (Ni-R) form, then transfers an electron to ferricyt-[c.sub.3] through the proximal, mesial and distal FeS clusters, from which the edge-to-edge distant to the nearest heme of the bound ferricyt-[c.sub.3] is only 6.4 [Angstrom], with [pi]-orbital rings of His238 and Phe247 located inbetween. (21) The resulting (Ni-C) form transfers the 2nd electron to ferricyt-[c.sub.3] to regenerate Ni-[SI.sub.1914] to complete the cycle. The [H.sup.+] produced can be released to the medium through a putative proton channel. (75,77) In the [H.sub.2]-evolution reaction, the inner circuit operating anticlockwise must be predominant, because p[H.sub.opt] [up arrow] lies below the p[K.sub.a]. Ni[SI.sub.1934] form receives electrons and [H.sup.+] successively to produce Ni[C.sub.1952], then Ni-[R.sub.1940], and finally releases [H.sub.2] to regenerate Ni-[SI.sub.1934] to complete the cycle.

In the isotopic exchange, only the right half semi-circuits are operating with both of Ni-SI forms, because the pH optimum for the exchange lies near the p[K.sub.a]. Both of the Ni-R forms are the molecular species which have properties compatible with the postulated [H.sub.2]-adduct of the enzyme, E[H.sub.a][H.sub.b], in Fig. 1, (6) where [H.sub.a] is suggested to be H bound to S-[Cys.sup.*], because the temperature factor of this residue is found to be unusually high compared to the other side chains in the protein by X-ray crystallography. (87) Since [Cys.sup.*] locates at the entrance to the putative proton-channel to the protein surface, (75,77) the bound H on S-[Cys.sup.*] must have higher exchange rate with medium [D.sup.+] than the bridging hydride between Ni and Fe, which is Hb. [NiFeSe]-hydrogenase has [Se.sup.+]-Sec instead of [S.sup.+]-[Cys.sup.*]. As selenol is more acidic than thiol, [Se.sup.+]-Sec would reduce the anionic character of the bridging hydride to make it more readily exchangeable with medium [D.sup.+] compared to [S.sup.+]-[Cys.sup.*], (73) i.e., [k.sub.b] of [NiFeSe]-enzyme would be higher than that of [NiFe]-enzyme, to make higher [v.sub.HH]/[v.sub.HD] ratios shown in Table 2. In fact, only 3 fold increase in [k.sub.b] is sufficient to account for the vhh/vhd ratio at pH 7.0 observed for [NiFeSe]-enzyme (Table 2), assuming the other kinetic constants unchanged. The proximal [4Fe-4S] cluster (not shown in Fig. 6), whose supporting residues are highly conserved among [NiFe]-hydrogenases (Fig. 2) is thought to be essential for the catalytic function. The catalytic mechanisms of [NiFe]-hydrogenase hitherto proposed70), (74,77,81,86,89,93) do not seem to aim at conforming to the results of kinetic studies (6)) schematically presented in Fig. 1.

Concluding remarks

The enzyme hydrogenase has been spotlighted since its discovery, because controlled reaction of the substrate of the enzyme, [H.sub.2], is possible only in the presence of catalyst, and the enzyme hydrogenase is expected to be a powerful tool to produce and utilize [H.sub.2] for future energy devices. Early achievements by studies on hydrogenases were the establishment of their electron carrier specificity and demonstration of heterolytic cleavage of [H.sub.2] on the enzyme. Our understanding on hydrogenases was remarkably widened and deepened since the elucidation of their three-dimensional structures by X-ray crystallography, and characterization of various states of the active site by spectroscopic studies such as EPR, FTIR etc. In this article, we surveyed the present knowledge on hydrogenases, and proposed the enzymatic mechanism by formulating the catalytic cycle running at the active site of the enzyme. This will provide a clue to design more efficient, [O.sub.2]-tolerant and stable catalyst by protein engineering, or to synthesize artificial catalysts for practical uses. Comprehensive reviews (7,75) are available for genetics, regulation or maturation of hydrogenases, not dealt with in this article.


We wish to express our gratitude to those who guided and encouraged us, especially to late Prof. Nobuo Tamiya and Prof. Hiroo Inokuchi, M.J.A., and to collaborators. Thanks are due to Prof. Tamio Yamakawa, M.J.A., who encouraged us to write this review article, and to Prof. Shigekazu Nagata, M.J.A., who communicated this paper at the meeting of the Japan Academy.


(1) Stephenson, M. and Stickland, L.H. (1931) Hydrogenase: a bacterial enzyme activating molecular hydrogen. I. The properties of the enzyme. Biochem. J. 25, 205-214.

(2) Stephenson, M. and Stickland, L.H. (1931) Hydrogenase II. The reduction of sulphate to sulphide by molecular hydrogen. Biochem. J. 25, 215-220.

(3) Krasna, A.I. and Rittenberg, D. (1954) The mechanism of action of the enzyme hydrogenase. J. Am. Chem. Soc. 76, 3015-3020.

(4) Rittenberg, D. and Krasna, A.I. (1955) Interaction of hydrogenase with hydrogen. Discuss. Faraday Soc. 20, 185-189.

(5) Tamiya, N. and Miller, S.L. (1963) Kinetic studies on hydrogenase. J. Biol. Chem. 238, 2194-2198.

(6) Yagi, T., Tsuda, M. and Inokuchi, H. (1973) Kinetic studies on hydrogenase. Parahydrogen-orthohydrogen conversion and hydrogen-deuterium exchange reactions. J. Biochem. 73, 1069-1081.

(7) Vignais, P.M. and Billoud, B. (2007) Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 107, 4206-4272.

(8) Kessler, E. (1973) Effect of anaerobiosis on photosynthesis reactions and nitrogen metabolism of algae with and without hydrogenase. Arch. Mikrobiol. 93,91-100.

(9) Lindmark, D.G. and Muller, M. (1967) Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J. Biol. Chem. 248, 7724-7728.

(10) Danovaro, R., Dell'Anno, A., Pusceddu, A., Gambi, C., Heiner, I. and Kristensen, R.M. (2010) The first metazoa living in permanently anoxic conditions. BMC Biol. 8, 30.

(11) Mortenson, L.E., Valentine, R.C. and Carnahan, J.E. (1962) An electron transport factor from Clostridium pasteurianum. Biochem. Biophys. Res. Commun. 7, 448-452.

(12) Bone, D.H., Bernstein, S. and Vishniac, W. (1963) Purification and some properties of different forms of hydrogen dehydrogenase. Biochim. Biophys. Acta 67, 581-588.

(13) Bone, D.H. (1963) Inhibitor, isotopic and kinetic studies on hydrogen dehydrogenase. Biochim. Biophys. Acta 67, 589-598.

(14) Tamiya, N., Yamaguchi, Y., Honya, M. and Yagi, T. (1966) The electron carrier specificities of hydrogenases of different organisms. Biochem. Biophys. Res. Commun. 22, 43-47.

(15) Yagi, T., Honya, M. and Tamiya, N. (1968) Purification and properties of hydrogenases of different origins. Biochim. Biophys. Acta 153, 699-705.

(16) Yagi, T. and Maruyama, K. (1971) Purification and properties of cytochrome [c.sub.3] of Desulfovibrio vulgaris, Miyazaki. Biochim. Biophys. Acta 243, 214-224.

(17) Yagi, T. (1984) Spectral and kinetic abnormality during the reduction of cytochrome [c.sub.3] catalyzed by hydrogenase with hydrogen. Biochim. Biophys. Acta 767, 288-294.

(18) Benosman, H., Asso, M., Bertrand, P., Yagi, T. and Gayda, J.P. (1989) EPR study of the redox interactions in cytochrome [c.sub.3] from Desulfovibrio vulgaris Miyazaki. Eur. J. Biochem. 182, 51-55.

(19) Bertrand, P., Asso, M., Mbarki, O., Camensuli, P., More, C. and Guigliarelli, B. (1994) Individual redox characteristics and kinetic properties of the hemes in cytochromes [c.sub.3]: New methods of investigation. Biochimie 76, 524-536.

(20) Yagi, T., Endo, A. and Tsuji, K. (1978) Properties of hydrogenase from particulate fraction of Desulfovibrio vulgaris. In Hydrogenases: Their Catalytic Activity, Structure and Function (eds. Schlegel, H.G. and Schneider, K.). Erich Goltze, Gottingen, pp. 107-124.

(21) Yahata, N., Saitoh, T., Takayama, Y., Ozawa, K., Ogata, H., Higuchi, Y. and Akutsu, H. (2006) Redox interaction of cytochrome [c.sub.3] with [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. Biochemistry 45, 1653-1662.

(22) Yagi, T. (1970) Solubilization, purification and properties of particulate hydrogenase from Desulfovibrio vulgaris. J. Biochem. 68, 649-657.

(23) Brugna, M., Giudici-Orticoni, M.T., Spinelli, S., Brown, K., Tegoni, M. and Bruschi, M. (1998) Kinetics and interaction studies between cytochrome [c.sub.3] and Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough. Proteins 33, 590-600.

(24) Teixeira, M., Moura, I., Xavier, A.V., Huynh, B.H., DerVartanian, D.V., Peck, H.D. Jr., LeGall, J. and Moura, J.J.G. (1985) Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas. J. Biol. Chem. 260, 8942-8950.

(25) Yamazaki, S., Tsai, L., Stadtman, T.C., Teshima, T., Nakaji, A. and Shiba, T. (1985) Stereo chemical studies of a selenium-containing hydrogenase from Methanococcus vannielii: determination of the absolute configuration of C-5 chirally labeled dihydro-8-hydroxy-5-deazaflavin cofactor. Proc. Natl. Acad. Sci. U.S.A. 82, 1364-1366.

(26) Klein, A.R., Fernandez, V.M. and Thauer, R.K. (1995) [H.sub.2]-forming [N.sup.5], [N.sup.10]-methylenetetrahydromethanopterin dehydrogenase: mechanism of [H.sub.2] formation analyzed using hydrogen isotopes. FEBS Lett. 368, 203-206.

(27) Liang, J. and Burris, R.H. (1988) Hydrogen burst associated with nitrogenase-catalyzed reactions. Proc. Natl. Acad. Sci. U.S.A. 85, 9446-9450.

(28) Menon, S. and Ragsdale, S.W. (1996) Unleashing hydrogenase activity in carbon monoxide dehydrogenase/acetyl-CoA synthase and pyruvate:ferredoxin oxidoreductase. Biochemistry 35, 15814-15821.

(29) Yang, K. and Metcalf, W.W. (2004) A new activity for an old enzyme: Escherichia coli bacterial alkaline phosphatase is a phosphite-dependent hydrogenase. Proc. Natl. Acad. Sci. U.S.A. 101, 7919-7924.

(30) Asso, M., Guigliarelli, B., Yagi, T. and Bertrand, P. (1992) EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas. Biochim. Biophys. Acta 1122,50-56.

(31) Voordouw, G. and Brenner, S. (1985) Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough). Eur. J. Biochem. 148, 515-520.

(32) Nicolet, Y., Piras, C., Legrand, P., Hatchikian, C.E. and Fontecilla-Camps, J.C. (1999) Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center. Structure 7, 13-23.

(33) Chen, J.S. and Mortenson, L.E. (1974) Purification and properties of hydrogenase from Clostridium pasteurianum W5. Biochim. Biophys. Acta 371, 283-298.

(34) Meuer, J., Kuettner, H.C., Zhang, J.K., Hedderich, R. and Metcalf, W.W. (2002) Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation. Proc. Natl. Acad. Sci. U.S.A. 99, 5632-5637.

(35) Voordouw, G., Menon, N.K., LeGall, J., Choi, E.S., Peck, H.D. Jr. and Przybyla, A.E. (1989) Analysis and comparison of nucleotide sequences encoding the genes for [NiFe] and [NiFeSe] hydrogenases from Desulfovibrio gigas and Desulfovibrio baculatus. J. Bacteriol. 171, 2894-2899.

(36) Zirngibl, C., Van Dongen, W., Schworer, B., Von Bunau, R., Richter, M., Klein, A. and Thauer, R.K. (1992) [H.sub.2]-forming methylenetetrahydro-methanopterin dehydrogenase, a novel type of hydrogenase without iron-sulfur clusters in methanogenic archaea. Eur. J. Biochem. 208, 511-520.

(37) Buurman, G., Shima, S. and Thauer, R.K. (2000) The metal-free hydrogenase from methanogenic archaea: evidence for a bound cofactor. FEBS Lett. 485, 200-204.

(38) Yagi, T., Tsuda, M., Mori, Y. and Inokuchi, H. (1969) Hydrogenase activity in the dry state. J. Am. Chem. Soc. 91, 2801.

(39) Kimura, K., Suzuki, A., Inokuchi, H. and Yagi, T. (1979) Hydrogenase activity in the dry state. Isotope exchange and reversible oxidoreduction of cytochrome [c.sub.3]. Biochim. Biophys. Acta 567, 96-105.

(40) Kimura, K., Nakahara, Y., Yagi, T. and Inokuchi, H. (1979) Electrical conduction of hemoprotein in the solid phase: anhydrous cytochrome [c.sub.3] film. J. Chem. Phys. 70, 3317-3323.

(41) Yagi, T., Inokuchi, H. and Kimura, K. (1983) Cytochrome [c.sub.3], a tetrahemoprotein electron carrier found in sulfate-reducing bacteria. Acc. Chem. Res. 16, 2-7.

(42) Jouanneau, Y., Kelley, B.C., Berlier, Y., Lespinat, P.A. and Vignais, P.M. (1980) Continuous monitoring, by mass spectrometry, of [H.sub.2] production and recycling in Rhodopseudomonas capsu lata. J. Bacteriol. 143, 628-636.

(43) Teixeira, M., Fauque, G., Moura, I., Lespinat, P.A., Berlier, Y., Prickril, B., Peck, H.D. Jr., Xavier, A.V., Le Gall, J. and Moura, J.J.G. (1987) Nickel-[iron-sulfur]-selenium- containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties. Eur. J. Biochem. 167, 47-58.

(44) Cournac, L., Guedeney, G., Peltier, G. and Vignais, P.M. (2004) Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex. J. Bacteriol. 186, 1737-1746.

(45) Gitlitz, P.H. and Krasna, A.I. (1975) Structural and catalytic properties of hydrogenase from Chromatium. Biochemistry 14, 2561-2568.

(46) Egerer, P., Gunther, H. and Simon, H. (1982) On the hydrogen-deuterium exchange reaction catalyzed by the soluble hydrogenase from Alcaligenes eutrophus H16 in the free and immobilized state. Biochim. Biophys. Acta 703, 149-157.

(47) Zorin, N.A., Dimon, B., Gagnon, J., Gaillard, J., Carrier, P. and Vignais, P.M. (1996) Inhibition by iodoacetamide and acetylene of the H-D-exchange reaction catalyzed by Thiocapsa roseopersicina hydrogenase. Eur. J. Biochem. 241, 675-681.

(48) Vignais, P.M., Henry, M.-F., Berlier, Y. and Lespinat, P.A. (1982) Effect of pH on the H-2H exchange, [H.sub.2] production and [H.sub.2] uptake catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans. Biochim. Biophys. Acta 681, 519-529.

(49) Lespinat, P.A., Berlier, Y., Fauque, G., Czechowski, B., Dimon, B. and Le Gall, J. (1986) The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria. Biochimie 68, 55-61.

(50) Schworer, B., Fernandez, V.M., Zirngibl, C. and Thauer, R.K. (1993) [H.sub.2]-forming [N.sup.5],[N.sup.10]- methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum. Studies of the catalytic mechanism of [H.sub.2] formation using hydrogen isotopes. Eur. J. Biochem. 212, 255-261.

(51) Saint-Martin, P., Lespinat, P.A., Fauque, G., Berlier, Y., LeGall, J., Moura, I., Teixeira, M., Xavier, A.V. and Moura, J.J.G. (1988) Hydrogen production and deuterium-proton exchange reactions catalyzed by Desulfovibrio nickel(II)-substituted rubredoxins. Proc. Natl. Acad. Sci. U.S.A. 85, 9378-9380.

(52) Kure, B., Matsumoto, T., Ichikawa, K., Fukuzumi, S., Higuchi, Y., Yagi, T. and Ogo, S. (2008) pH-Dependent isotope exchange and hydrogenation catalysed by water-soluble NiRu complexes as functional models for [NiFe]hydrogenases. Dalton Trans. 2008, 4747-4755.

(53) Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S. and Inokuchi, H. (1976) Properties of purified hydrogenase from the particulate fraction of Desulfovibrio vulgaris, Miyazaki. J. Biochem. 79, 661-671.

(54) Hatchikian, E.C., Bruschi, M., Le Jall, J., Forget, N. and Bovier-Lapierre, N. (1978) Characterization of the periplasmic hydrogenase from Desulfovibrio gigas. Biochem. Biophys. Res. Commun. 82, 451-461.

(55) Dross, F., Geisler, V., Lenger, R., Theis, F., Krafft, T., Fahrenholz, F., Kojro, E., Duchene, A., Tripier, D., Juvenal, K. and Kroger, A. (1992) The quinone-reactive Ni/Fe-hydrogenase of Wolinella succinogenes. Eur. J. Biochem. 206, 93-102 [Erratum (1993) Eur. J. Biochem. 214, 949-950].

(56) Abken, H.J., Tietze, M., Brodersen, J., Baumer, S., Beifuss, U. and Deppenmeier, U. (1998) Isolation and characterization of methanophenazine and function of phenazines in membrane-bound electron transport of Methanosarcina mazei Go1. J. Bacteriol. 180, 2027-2032.

(57) Deppenmeier, U., Blaut, M., Lentes, S., Herzberg, C. and Gottschalk, G. (1995) Analysis of the vhoGAC and vhtGAC operons from Methanosarcina mazei strain Goe1, both encoding a membrane-bound hydrogenase and a cytochrome b. Eur. J. Biochem. 227, 261-269.

(58) Alex, L.A., Reeve, J.N., Orme-Johnson, W.H. and Walsh, C.T. (1990) Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum [DELTA]H. Biochemistry 29, 7237-7244.

(59) Burgdorf, T., van der Linden, E., Bernhard, M., Yin, Q.Y., Back, J.W., Hartog, A.F., Muijsers, A.O., de Koster, C.G., Albracht, S.P.J. and Friedrich, B. (2005) The soluble [NAD.sup.+]-reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH. J. Bacteriol. 187, 3122-3132.

(60) Shomura, Y., Yoon, K.S., Nishihara, H. and Higuchi, Y. (2011) Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membranebound [NiFe]-hydrogenase. Nature 479, 253-256.

(61) Sargent, F., Bogsch, E.G., Stanley, N.R., Wexler, M., Robinson, C., Berks, B.C. and Palmer, T. (1998) Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J. 17, 3640-3650.

(62) Weiner, J.H., Bilous, P.T., Shaw, G.M., Lubitz, S.P., Frost, L., Thomas, G.H., Cole, J.A. and Turner, R.J. (1998) A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell 93, 93-101.

(63) Meyer, J. and Gagnon, J. (1991) Primary structure of hydrogenase I from Clostridium pasteurianum. Biochemistry 30, 9697-9704.

(64) Atta, M. and Meyer, J. (2000) Characterization of the gene encoding the [Fe]-hydrogenase from Megasphaera elsdenii. Biochim. Biophys. Acta 1476, 368-371.

(65) Schut, G.J. and Adams, M.W.W. (2009) The iron hydrogenase of Thermotoga maritima utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen production. J. Bacteriol. 191, 4451-4457.

(66) de Luca, G., de Philip, P., Rousset, M., Belaich, J.P. and Dermoun, Z. (1998) The NADP-reducing hydrogenase of Desulfovibrio fructosovorans: Evidence for a native complex with hydrogen-dependent methyl-viologen-reducing activity. Biochem. Biophys. Res. Commun. 248, 591-596.

67) Hartmann, G.C., Klein, A.R., Linder, M. and Thauer, R.K. (1996) Purification, properties and primary structure of [H.sub.2]-forming [N.sup.5],[N.sup.10]- methylenetetrahydromethanopterin dehydrogenase from Methanococcus thermolithotrophicus. Arch. Microbiol. 165, 187-193.

(68) Shima, S., Pilak, O., Vogt, S., Schick, M., Stagni, M.S., Meyer-Klaucke, W., Warkentin, E., Thauer, R.K. and Ermler, U. (2008) The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site. Science 321, 572-575.

(69) Volbeda, A., Charon, M.H., Piras, C., Hatchikian, E.C., Frey, M. and Fontecilla-Camps, J.C. (1995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580-587.

(70) Volbeda, A., Garcin, E., Piras, C., de Lacey, A.L., Fernandez, V.M., Hatchikian, E.C., Frey, M. and Fontecilla-Camps, J.C. (1996) Structure of the [NiFe] hydrogenase active site: evidence for biologically uncommon Fe ligands. J. Am. Chem. Soc. 118, 12989-12996.

(71) Higuchi, Y., Yagi, T. and Yasuoka, N. (1997) Unusual ligand structure in Ni-Fe active center and an additional Mg site in hydrogenase revealed by high resolution X-ray structure analysis. Structure 5, 1671-1680.

(72) Peters, J.W., Lanzilotta, W.N., Lemon, B.J. and Seefeldt, L.C. (1998) X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution. Science 282, 1853-1858 [Erratum: (1998) Science 283, 35].

(73) Garcin, E., Vernede, X., Hatchikian, E.C., Volbeda, A., Frey, M. and Fontecilla-Camps, J.C. (1999) The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center. Structure 7, 557-566.

(74) De Lacey, A.L., Hatchikian, E.C., Volbeda, A., Frey, M., Fontecilla-Camps, J.C. and Fernandez, V.M. (1997) Infrared-spectroelectrochemical characterization of the [NiFe] hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc. 119, 7181-7189.

(75) Fontecilla-Camps, J.C., Volbeda, A., Cavazza, C. and Nicolet, Y. (2007) Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chem. Rev. 107, 4273-4303.

(76) Marques, M.C., Coelho, R., De Lacey, A.L., Pereira, I.A.C. and Matias, P.M. (2010) The three-dimensional structure of [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough: a hydrogenase without a bridging ligand in the active site in its oxidised, "as-isolated" state. J. Mol. Biol. 396, 893-907.

(77) Ogata, H., Lubitz, W. and Higuchi, Y. (2009) [NiFe] hydrogenases: structural and spectroscopic studies of the reaction mechanism. Dalton Trans. 7, 7577-7587.

(78) Volbeda, A., Martin, L., Cavazza, C., Matho, M., Faber, B.W., Roseboom, W., Albracht, S.P.J., Garcin, E., Rousset, M. and Fontecilla-Camps, J.C. (2005) Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases. J. Biol. Inorg. Chem. 10, 239-249 [Erratum: (2005) J. Biol. Inorg. Chem. 10, 591].

(79) Carepo, M., Tierney, D.L., Brondino, C.D., Yang, T.C., Pamplona, A., Telser, J., Moura, I., Moura, J.J.G. and Hoffman, B.M. (2002) 17O ENDOR detection of a solvent-derived Ni-(OHx)-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas. J. Am. Chem. Soc. 124, 281-286.

(80) Higuchi, Y. and Yagi, T. (1999) Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen. Biochem. Biophys. Res. Commun. 255, 295-299.

(81) Higuchi, Y., Ogata, H., Miki, K., Yasuoka, N. and Yagi, T. (1999) Removal of the bridging ligand atom at the Ni-Fe active site of [NiFe] hydrogenase upon reduction with [H.sub.2], as revealed by X-ray structure analysis at 1.4 [Angstrom] resolution. Structure 7, 549-556.

(82) van Gastel, M., Stein, M., Brecht, M., Schroder, O., Lendzian, F., Bittl, R., Ogata, H., Higuchi, Y. and Lubitz, W. (2006) A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. J. Biol. Inorg. Chem. 11, 41-51.

(83) Ogata, H., Hirota, S., Nakahara, A., Komori, H., Shibata, N., Kato, T., Kano, K. and Higuchi, Y. (2005) Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state. Structure 13, 1635-1642.

(84) Pierik, A.J., Roseboom, W., Happe, R.P., Bagley, K.A. and Albracht, S.P.J. (1999) Carbon monoxide and cyanide as intrinsic ligands to iron in the active site of [NiFe]-hydrogenases. J. Biol. Chem. 274, 3331-3337.

(85) Schenk, W.A. (1987) Sulfur oxides as ligands in coordination compounds. Angew. Chem. Int. Ed. Engl. 26, 98-109.

(86) Higuchi, Y., Toujou, F., Tsukamoto, K. and Yagi, T. (2000) The presence of a SO molecule in [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki as detected by mass spectrometry. J. Inorg. Biochem. 80, 205-211.

(87) Ogata, H., Mizoguchi, Y., Mizuno, N., Miki, K., Adachi, S., Yasuoka, N., Yagi, T., Yamauchi, O., Hirota, S. and Higuchi, Y. (2002) Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen. J. Am. Chem. Soc. 124, 11628-11635.

(88) Nishihara, H., Miyashita, Y., Aoyama, K., Kodama, T., Igarashi, Y. and Takamura, Y. (1997) Characterization of an extremely thermophilic and oxygen-stable membrane-bound hydrogenase from a marine hydrogen-oxidizing bacterium Hydrogenovibrio marinus. Biochem. Biophys. Res. Commun. 232, 766-770.

(89) Goris, T., Wait, A.F., Saggu, M., Fritsch, J., Heidary, N., Stein, M., Zebger, I., Lendzian, F., Armstrong, F.A., Friedrich, B. and Lenz, O. (2011) A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase. Nat. Chem. Biol. 7, 310-318 [Erratum: (2011) Nat. Chem. Biol. 7, 648].

(90) Fritsch, J., Scheerer, P., Frielingsdorf, S., Kroschinsky, S., Friedrich, B., Lenz, O. and Spahn, C.M. (2011) The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 479, 249-252.

(91) Nicolet, Y., de Lacey, A.L., Vernede, X., Fernandez, V.M., Hatchikian, E.C. and Fontecilla-Camps, J.C. (2001) Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans. J. Am. Chem. Soc. 123, 1596-1601.

(92) Teixeira, M., Moura, I., Xavier, A.V., Moura, J.J.G., LeGall, J., DerVartanian, D.V., Peck, H.D. Jr. and Huynh, B.H. (1989) Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. Mossbauer and EPR characterization of the metal centers. J. Biol. Chem. 264, 16435-16450.

(93) Dole, F., Fournel, A., Magro, V., Hatchikian, E.C., Bertrand, P. and Guigliarelli, B. (1997) Nature and electronic structure of the Ni-X dinuclear center of Desulfovibrio gigas hydrogenase. Implications for the enzymatic mechanism. Biochemistry 36, 7847-7854.

(94) Yagi, T., Kimura, K. and Inokuchi, H. (1985) Analysis of the active center of hydrogenase from Desulfovibrio vulgaris Miyazaki by magnetic measurements. J. Biochem. 97, 181-187.

(95) Trofanchuk, O., Stein, M., Gessner, C., Lendzian, F., Higuchi, Y. and Lubitz, W. (2000) Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. J. Biol. Inorg. Chem. 5, 36-44.

(96) Yagi, T., Goto, M., Nakano, K., Kimura, K. and Inokuchi, H. (1975) A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method. J. Biochem. 78, 443-454.

(97) Yagi, T. and Higuchi, Y. (2012) Catalytic cycle of cytochrome-[c.sub.3] hydrogenase, a [NiFe]-hydroge nase. Seikagaku 84, Abstract No. 2T02-05.

(98) De Gioia, L., Fantucci, P., Guigliarelli, B. and Bertrand, P. (1999) Ab initio investigation of the structural and electronic differences between active-site models of [NiFe] and [NiFeSe] hydrogenases. Int. J. Quantum Chem. 73, 187-195.

(99) Foerster, S., Stein, M., Brecht, M., Ogata, H., Higuchi, Y. and Lubitz, W. (2003) Single crystal EPR studies of the reduced active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. J. Am. Chem. Soc. 125, 83-93.

(100) Foerster, S., van Gastel, M., Brecht, M. and Lubitz, W. (2005) An orientation-selected ENDOR and HYSCORE study of the Ni-C active state of Desulfovibrio vulgaris Miyazaki F hydrogenase. J. Biol. Inorg. Chem. 10, 51-62.

(Communicated by Shigekazu NAGATA, M.J.A.)

(Received Sep. 20, 2012; accepted Nov. 1, 2012)


Tatsuhiko Yagi was born in 1933. He graduated from the Department of Chemistry, Faculty of Science, University of Tokyo in 1955. Discovery of carbon monoxide dehydrogenase was his first achievement as a graduate student of the same university. He moved to the Institute for Hard Tissue Research, Tokyo Medical and Dental University, as an assistant of Prof. Nobuo Tamiya in 1958. As a Fulbrigher he worked in the laboratory of Prof. Andrew A. Benson at the Pennsylvania State University, where he contributed to correct the structure of sulfolipid, which had been erroneously described. On returning to Japan in 1962, he started his life-work on hydrogenase with Prof. Tamiya. After obtaining the PhD degree on science, he moved to Shizuoka University in 1966, and was promoted to professor in 1972. His accomplishments there include, in addition to the studies on hydrogenase, elucidation of structure and function of bacterial electron carrier proteins such as cytochrome [c.sub.3], cytochrome c-553, ferredoxins and rubredoxin, discovery of multiheme high-molecular- weight cytochrome c, practice of an enzymatic electric cell for the activity assay, discovery of 1-methoxyPMS as a versatile photostable electron mediator, etc. He helped Prof. Tamiya by reinforcing his hypothesis on evolution. He published many biochemical textbooks with him, and edited the Enzyme Handbook and an encyclopedia of compounds to search for enzymes. He is a councilor of the Japanese Biochemical Society, an emeritus member of the American Chemical Society and a member of the Chemical Society of Japan.


Yoshiki Higuchi was born in 1956 and started his research career in 1979 under the guidance of Prof. Masao Kakudo at Institute for Protein Research, Osaka University, and obtained his PhD degree for his X-ray crystallographic study on cytochrome [c.sub.3] in 1984. He was appointed as an Assistant Professor of Himeji Institute of Technology in 1985, moved to Kyoto University in 1995 as an Associate Professor of Graduate School of Science. He moved to Himeji Institute of Technology, as a Professor at the Graduate School of Science in 2002, and is currently a Professor at the Graduate School of Life Science, University of Hyogo. He has been involved in the studies on the structure and function of a [NiFe] hydrogenase from sulfate-reducing bacterium. He developed the purification and crystallization procedures of the hydrogenase and obtained the first single crystals in 1987, and succeeded in high resolution X-ray structural analysis of various forms with diverse activities of the same [NiFe] hydrogenase, a thorough investigation on a single object. At the same time, he successfully solved structure-function relationship on bacterial multi-copper oxidase, and other enzymes. He also solved the structure of the bacterial translational release factor, RF-3, complexed with a stringent factor, ppGpp. He received the Award of the Crystallographic Society of Japan in 1999. At present, he is involved in structural chemistry on hydrogenases including those from other sources, as well as on protein-protein interactions in the cells of higher organisms. He is a member of the Japanese Biochemical Society, the Chemical Society of Japan, the Protein Science Society of Japan, the Crystallographic Society of Japan, etc.

By Tatsuhiko YAGI [*1], ([dagger]) and Yoshiki HIGUCHI [*2]

[*1] Shizuoka University, Shizuoka, Japan.

[*2] Graduate School of Life Science, University of Hyogo, Hyogo, Japan.

([dagger]) Correspondence should be addressed: T. Yagi, 1664-33, Kita, Aoi-ku, Shizuoka 420-0961 (e-mail:

Table 1. Hydrogenases registered by IUBMB

EC number           Name of the Enzyme (a)

EC (b)     [NAD.sup.+]-reducing hydrogenase (c) or
                      Hydrogen dehydrogenase
EC         [NADP.sup.+]-reducing hydrogenase (c) or
                      Hydrogen dehydrogenase ([NADPD.sup.+])

EC (d)     Bifurcating hydrogenase (c) or
                      Hydrogenase ([NAD.sup.+], ferredoxin)
EC         Cytochrome-[c.sub.3] hydrogenase
EC (e)     Quinone-reducing hydrogenasec) or
                      Hydrogen:quinone oxidoreductase
EC         Ferredoxin hydrogenase
EC (f)    [F.sub.420]-reducing hydrogenasec) or
                      Coenzyme [F.sub.420] hydrogenase
EC (g)    5,10-Methenyltetrahydromethanopterin
                      hydrogenase or [H.sub.2]-forming
                      dehydrogenase (c)
EC (e)    Methanophenazine hydrogenase (c)
EC (h)    Hydrogenase (acceptor) or
                      Hydrogen:acceptor oxidoreductase

(a) Hydrogenase is named, in principle, as an enzyme catalyzing the
uptake of [H.sub.2] to reduce its electron acceptor, even if the
evolution of [H.sub.2] is relevant in vivo. The physiological
electron acceptor of the enzyme can be read by the name of the
enzyme. (b)) Addition of H occurs from the si-face of C-4 of the
pyridine ring of [NAD.sup.+]. (13) (c) Names found in literature,
but not accepted by IUBMB. (d) Catalyzes the production of [H.sub.2]
only when both of NADH and reduced Fd are present. (e) Uptake-
hydrogenase, i.e., the enzyme does not catalyze production of
[H.sub.2] from the reduced electron carrier. (f) Addition of H
occurs from the si-face of C-5 of the deazaflavin ring. (25)
g)Addition of H occurs from the re-face of C between N-5 and N-10 of
5,10-methenyltetrahydromethanopterin. (26) In the absence of 5,10-
methenyltetrahydromethanopterin, this enzyme does not catalyze H-D
exchange reaction or the reduction of artificial electron acceptors.
(h) Hydrogenases acting only on artificial electron carriers. They
will be given proper names when their respective electron carriers
are specified.

Table 2. Summary of the isotope exchange and
conversion reactions by hydrogenases

Hydrogenase                                  /[v.sub.ex] (pD)

[NiFe] (a)      D. vulgaris                  (with cyt-[c.sub.3])
                Miyazaki 6)                  (without cyt-[c.sub.3])

[NiFe] (a,f)    D. gigas (43)

[NiFe] (a)      D. fructosovorans (44)
[NiFe] (a)      Chromatium (45)
[NiFe] (a)      P. vulgaris (3-5)
[NiFe] (a)      [NAD.sup.+]-reducing
                Ralstonia eutropha (46)

[NiFe]          Rhod. Capsulata (42)

[NiFe] (a,i)    Thiocapsa
                roseopersicina (47)

[NiFe] (j)      P. denitrificans (48)

[NiFeSe]        D. baculatum (43)

[FeFe] (a,b)    C. pasteurianum (5)

[FeFe] (a,f)    D. vulgaris
                Hildenborough (49)

[Fe] (k)        M.
                  thermoautotrophicum (50)

cf.             Ni-substituted
                  rubredoxin (51)
                Ni-Ru bimetallic
                  complex (52)

Hydrogenase                                  /[v.sub.ex] (pD)

[NiFe] (a)      D. vulgaris                  0.7, (b) 0.2 (c) (7.4)
                Miyazaki 6)                  n.d., (b,e) 0.5 (c) (7.4)

[NiFe] (a,f)    D. gigas (43)

[NiFe] (a)      D. fructosovorans (44)
[NiFe] (a)      Chromatium (45)
[NiFe] (a)      P. vulgaris (3-5)            <0.1 (7.0)
[NiFe] (a)      [NAD.sup.+]-reducing
                Ralstonia eutropha (46)

[NiFe]          Rhod. Capsulata (42)

[NiFe] (a,i)    Thiocapsa
                roseopersicina (47)

[NiFe] (j)      P. denitrificans (48)

[NiFeSe]        D. baculatum (43)

[FeFe] (a,b)    C. pasteurianum (5)          <0.1 (7.4)

[FeFe] (a,f)    D. vulgaris
                Hildenborough (49)

[Fe] (k)        M.
                  thermoautotrophicum (50)

cf.             Ni-substituted
                  rubredoxin (51)
                Ni-Ru bimetallic
                  complex (52)

Hydrogenase                                  (pD)

[NiFe] (a)      D. vulgaris                  0.20, b) 1.5 c) (7.4) (d)
                Miyazaki 6)                  0.2, b) 1.1 c) (7.4) (d)

[NiFe] (a,f)    D. gigas (43)

[NiFe] (a)      D. fructosovorans (44)
[NiFe] (a)      Chromatium (45)              0.40 (6.0)
[NiFe] (a)      P. vulgaris (3-5)            0.19b) (7.4)
[NiFe] (a)      [NAD.sup.+]-reducing
                Ralstonia eutropha (46)

[NiFe]          Rhod. Capsulata (42)         0.78g) (8.0)

[NiFe] (a,i)    Thiocapsa
                roseopersicina (47)

[NiFe] (j)      P. denitrificans (48)        1.77 (4.5) (d) 1.27 (8.3)

[NiFeSe]        D. baculatum (43)

[FeFe] (a,b)    C. pasteurianum (5)          0.18 (5.5) 0.34 (6.5)
                                               0.57 (7.4)
                                             0.47 (8.0) 0.27 (8.4)
                                               (d) 0.12 (9.6)
[FeFe] (a,f)    D. vulgaris
                Hildenborough (49)

[Fe] (k)        M.
                  thermoautotrophicum (50)

cf.             Ni-substituted               1.45 (6.8)
                  rubredoxin (51)
                Ni-Ru bimetallic             3.4 (4.0) at 333K
                  complex (52)

Hydrogenase                                  (pH)

[NiFe] (a)      D. vulgaris                  0.40, (b) 3.6 (c) (7.0)
                Miyazaki 6)                    (d)
                                             0.40, (b) 1.6 (c) (7.0)
[NiFe] (a,f)    D. gigas (43)                0.32 (5.0) 0.32 (6.6)
                                               0.34 (8.0) d)
                                             0.48 (9.0) 0.55 (9.5)
                                               0.32 (11.0)
[NiFe] (a)      D. fructosovorans (44)       0.28 (7.2)
[NiFe] (a)      Chromatium (45)
[NiFe] (a)      P. vulgaris (3-5)
[NiFe] (a)      [NAD.sup.+]-reducing         0.10 (5.5) 0.20 (6.1)
                Ralstonia eutropha (46)        0.22 (6.5)
                                             0.30 (7.3) d) 0.40 (8.2)
                                               0.30 (9.0)
[NiFe]          Rhod. Capsulata (42)         2.44 g) (8.0), 1.97 h)
                                             (8.0) (d)
[NiFe] (a,i)    Thiocapsa                    0.22 (4.0) 0.30 (5.5) d)
                roseopersicina (47)            0.41 (6.5)
                                             0.47 (8.0) 0.44 (9.0)
                                               0.30 (9.5)
[NiFe] (j)      P. denitrificans (48)        2.30 (4.5)d) 1.92 (7.0)
                                               1.68 (8.3)
[NiFeSe]        D. baculatum (43)            0.19 (2.6) 0.51 (3.5)
  (a,f)                                        1.01 (4.5) (d)
                                             1.20 (5.5) 1.29 (7.0)
                                               1.43 (7.6)
[FeFe] (a,b)    C. pasteurianum (5)

[FeFe] (a,f)    D. vulgaris                  0.10 (3.0) 0.27 (4.0)
                Hildenborough (49)             0.34 (4.8) (d)
                                             0.62 (6.0) 0.52 (7.0)
                                               0.40 (8.0)
[Fe] (k)        M.                           1.0 (5.7) 1.0 (6.7-7.0)
                  thermoautotrophicum (50)     (d) 1.1 (7.5)

cf.             Ni-substituted
                  rubredoxin (51)
                Ni-Ru bimetallic             7.0 (3.9) at 333K
                  complex (52)

(a) Purified or partially purified enzyme. (b) Enzyme concentration
extrapolated to 0. (c) High enzyme concentration (about 50 nmol
[L.sup.-1]). (d) Optimum pD or pH for the exchange reaction. (e) Not
determined due to sluggish reaction rate. (f) Low enzyme concentration
(about 2 nmol [L.sup.-1]). (g) Chromatophores. (h) whole cells. (i)
Enzyme about 6.6 nmol [L.sup.-1]. (j) Membrane particles. (k) In the
presence of its electron acceptor,
COPYRIGHT 2013 The Japan Academy
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2013 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Yagi, Tatsuhiko; Higuchi, Yoshiki
Publication:Japan Academy Proceedings Series B: Physical and Biological Sciences
Article Type:Report
Geographic Code:9JAPA
Date:Jan 1, 2013
Previous Article:Visualization of chemical reaction dynamics: toward understanding complex polyatomic reactions.
Next Article:Evolution of basic equations for nearshore wave field.

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters