Soluble Hydrogenase Research Articles

NADH‐Induced Changes of the Nickel Coordination within the Active Site of the Soluble Hydrogenase from Alcaligenes eutrophus: XAFS Investigations on Three States Distinguishable by EPR Spectroscopy

NADH‐Induced Changes of the Nickel Coordination within the Active Site of the Soluble Hydrogenase from <i>Alcaligenes eutrophus</i>: XAFS Investigations on Three States Distinguishable by EPR Spectroscopy

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (betadelta) and an NADH:acceptor oxidoreductase (alphagamma)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed.

Alcaligenes Eutrophus Cells Containing Hydrogenase, a Coenzyme Regenerating Catalyst for Nadh-Dependent Oxidoreductases

Alcaligenes eutrophus cells containing a soluble NAD-dependent hydrogenase was evaluated as a NADH regenerating catalyst. In the presence of hydrogen and permeabilized A. eutrophus cells, NADH regeneration was achieved, and reductions catalyzed by horse liver alcohol de-hydrogenase and lactate dehydrogenase proceeded to 90–99% conversion. The operational stability of the hydrogenase was improved by the addition of reducing agents such as dithio threitol and by immobilization of the cells in calcium alginate.

Structure of the Ni Sites in Hydrogenases by X-ray Absorption Spectroscopy. Species Variation and the Effects of Redox Poise

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD+-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587) is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. The nickel K-edge energies shift 0.9−1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (∼0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent intermediate redox level (SI). Analysis of the XANES features assigned to 1s → 3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The smallest edge energy shift is observed for the T. roseopersicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample of A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at ∼2.2 Å. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3−4 N,O-donor atoms at 2.06(2) Å, with contributions from 2−3 S-donor ligands at 2.35(2) Å. This changes upon reduction to a more typical nickel site composed of ∼4 S-donor ligands at a Ni−S distance of 2.19(2) Å. Evidence for the presence of atoms in the 2.4−2.9 Å distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to distinguish between S and Fe scattering atoms at this distance, and by the potential presence of both S and another metal atom at similar distances. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of the D. gigas enzyme.

Preparation of Solutions of Dissolved Hydrogen and Oxygen Gases Using an Anaerobic Chamber

The oxygen-tolerant, NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising catalyst for cofactor regeneration in enzyme-catalysed reduction processes. The technical use of the isolated enzyme, however, is limited by its relatively low stability under operational conditions such as agitation, elevated temperature or addition of co-solvents. The maximum half-life at a reaction temperature of 35 °C and pH 8.0 was only 5.3 h. In order to enhance the stability of the enzyme, it was immobilised onto the anionic resin Amberlite™ FPA54. At an immobilisation yield of 93.4% for adsorptive and 100% for covalent attachment, corresponding activities of 48.9 and 39.3%, respectively, were obtained. Covalent binding always yielded superior stabilisation. At elevated temperature and under agitation, stabilisation was further increased by modification of the covalently bound SH with methoxy-poly(ethylene) glycol (mPEG). A comparable effect was not achieved when SH modification was performed before immobilisation. In stationary aqueous solution, half-lives of up to 161 h at 25 °C and 32 h at 35 °C were obtained. In presence of the technically relevant co-solvents DMSO, DMF, 2-propanol and [EMIM][EtSO4] half-lives of 14–29 h can now be achieved.

NADH:ferredoxin reductase and NAD-reducing hydrogenase activities in Hydrogenobacter thermophilus strain TK-6

NADH:ferredoxin reductase (EC 1.18.1.3) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifically with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.

Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product

Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.

The NAD-linked soluble hydrogenase from Alcaligenes eutrophus H16: detection and characterization of EPR signals deriving from nickel and flavin

In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme was either reduced with the natural electron carrier NADH (5–10 mM) or carefully titrated with sodium dithionite to an intermediate, narrow redox potential range (–280 to –350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at 80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments into three distinct signals: (1) the "classical" Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal (Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with 61Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved 61Ni hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials determined at pH 7.0 for the described redox components were: for FMN –170 mV (midpoint potential, Em, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (Em for disappearance); for the Ni centre (Ni-C), –290 mV (Em for appearance), –305 mV (signal intensity maximum) and –325 mV (Em for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01.

The [Ni-Fe] hydrogenase from the thermophilic bacterium Acetomicrobium flavidum.

Biochemical analysis of the soluble hydrogenase from the thermophilic organism Acetomicrobium flavidum revealed that the enzyme is an alpha 2 beta 2 tetramer, with the alpha and beta subunits having a molecular mass of 50 kDa and 25 kDa, respectively. The most important biochemical properties of the enzyme are a specific activity of 77 mumol min-1 (mg protein)-1, a Km for methylviologen of 0.2 mM, a pH optimum of 7.5 and a T50 of about 70 degrees C. In addition, the enzyme is remarkably stable to oxygen inactivation, retaining full activity after 24 h exposure to air. By using oligodeoxynucleotides designed on the basis of the N-terminal sequences of the two subunits, the corresponding genes have been isolated and sequenced. When compared to the other hydrogenases so far characterized, the A. flavidum hydrogenase appears to be a typical [Ni-Fe] enzyme. The hydrogenase was expressed in Escherichia coli at high levels in a soluble form but it was not active. The analysis of the recombinant large subunit showed that it was not post-translationally processed at its C-terminus.

Kinetic parameters for hydrogen evolution by the NAD-linked hydrogenase of Alcaligenes eutrophus

The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H2-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K m 0.7 mM) the hydrogenase was most active at a redox potential of −560 mV and exhibited a pH optimum of 7.0. The K m for protons, the second substrate for H2 evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H2 is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H2 evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.

Nickel XAFS investigations of the soluble NiFe hydrogenase from Alcaligenes eutrophus

Nickel XAFS investigations of the soluble NiFe hydrogenase from Alcaligenes eutrophus

Kinetic Mechanism Studies of the Soluble Hydrogenase from Alcaligenes eutrophus H16

Purified soluble hydrogenase (H2:NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus was activated to high specific activities by flushing the enzyme consecutively with N2 and H2 and then adding substoichiometric quantities of NADH. H2-dependent NAD+ reduction activities ≥ 110 μmol NADH formed/min/mg protein at pH 8.0 and 30°C were obtained which were stable for several hours at 4°C. Kinetic studies were conducted anaerobically using activated enzyme for the purpose of evaluating the potential of using hydrogenase to enhance decompression of mammals breathing H2/O2 mixtures under hyperbaric conditions (i.e., at ambient pressures greater than 1 atm). Using nonlinear curve fitting of the kinetic data, it was found that H2 and NAD+ bind hydrogenase via a ping pong bi bi mechanism with Km values (±SE) of 11 ± 0.9 and 138 ± 11 μM, respectively, at 30°C and pH 8.0. Sodium ions were found to reversibly inhibit hydrogenase via a dead-end type of inhibition in which two catalytic forms of the enzyme bind Na+ with dissociation constants calculated to be 8.3 ± 1.2 and 49.8 ± 11.5 mM. In the absence of NaCl, maximum NAD+ reduction activity was measured at pH 8.3 at 30 and 37°C. In the presence of 50 mM NaCl, inhibition was observed primarily at alkaline pH, and at assay pH values ≤ 7.0, little or no difference was observed in activity in the presence or absence of 50 mM NaCl at a given temperature. Least squares analyses of the kinetic data indicated that substrate inhibition by H2 occurs at high substrate concentrations (Ki = 1.46 ± 0.64 mM), which would become a significant influence on enzyme catalytic activity at hyperbaric levels of H2.

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

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

Characterization and purification of a hydrogenase from the eukaryotic green alga Scenedesmus obliquus

Several catalytic properties of the hydrogenase from Scenedesmus obliquus have been examined to optimize the purification conditions. The K m -value for H 2-evolution in the presence of the most effective electron mediator methylviologen is 0.66 mM. The pH-optimum is 6.3, the temperature-optimum is 50°C and the energy of activation is 38.4 ± 2 kJ·mol −1. The soluble hydrogenase from the green alga, Scenedesmus obliquus, was purified 1290-fold to homogeneity. The enzyme consists of two subunits with molecular masses of 55 kDa and 36 kDa. The molecular weight of the native enzyme, determined by gel filtration, is 150 ± 5 kDa.

Homology of the NifS family of proteins to a new class of pyridoxal phosphate-dependent enzymes

Iterative profile sequence analysis reveals a remote homology of peroxisomal serine-pyruvate aminotransferases from mammals to the small subunit of soluble hydrogenases from cyanobacteria, an isopenicillin N epimerase, the NifS gene products from bacteria and yeast, and the phosphoserine aminotransferase family. All members of this new class whose function is known are pyridoxal phosphate-dependent enzymes, yet they have distinct catalytic activities. Upon alignment, a lysine around position 200 remains invariant and is predicted to be the pyridoxal phosphate-binding residue. Based on the detected homology, it is predicted that NifS has also a pyridoxal phosphate-dependent serine (or related) aminotransferase function associated with nitrogen economy and/or protection during nitrogen fixation.

Possible evolutionary relationship between mammalian alanine:glyoxylate aminotransferase 1 and the 42-kD subunit of cyanobacterial soluble hydrogenase.

Using the sequence comparison programs TFASTA / FASTA ( Pearson and Lipman 1988) to search the GenBank/EMBL DNA data bases, we have discovered that there is considerable amino acid sequence identity between mammalian alanine: glyoxylate aminotransferase 1 (AGT; E.C.2.6.1.44) and the 42-kD tritium-exchange subunit of cyanobacterial soluble hydrogenase (TES; E.C. 1.12.-.-/ 1.18.-.-) . Over a 36 l-amino-acid region (residues 24-384) of human liver AGT (Takada et al. 1990) and a 355amino-acid region (residues 6-360) of Anabaena cylindrica TES (Ewart et al. 1990)) 110 amino acids were found to be identical, and 90 were found to be similar (using the criteria advocated by Barker and Dayhoff 1972) (see fig. 1). When gaps are counted as mismatches, the level of sequence identity is 30. I%, and similarity is 54.8%. Similar results were obtained when comparisons were made between TES from Anabaena or Synechococcus (Van der Oost et al. 1989) and the AGT sequences obtained from rat ( Oda et al. 1987 ), marmoset, and rabbit (Purdue et al. 1992). Eightytwo (23.7%) amino acids are absolutely conserved in all six proteins (i.e., two TESS and four AGTs) (see fig. 1). In some regions there is considerably greater match between Anabaena TES and human AGT. For example, TES residues 66-98 and AGT residues 85-117 show 17/33 (51.5%) identity and 27/33 (81.8%) similarity. Two regions (TES 137-148/AGT 153-164, and TES 164-175/AGT 179-190) show 7 / 12 ( 58.3% ) amino acid identity and 12/ 12 ( 100% ) similarity. Whether these regions represent equivalent functional domains has yet to be determined. AGT, like all aminotransferases, requires pyridoxal phosphate as a cofactor. The binding site for this cofactor has been suggested to be Lys-209 (Oda et al. 1987). There is no evidence that hydrogenases require pyridoxal phosphate. Therefore, it is surprising to find that, not only is this Lys absolutely conserved in both TES sequences, in addition to all four AGT sequences, but the four preceding amino acids are conserved as well. However, this SerGlySerGlnLys sequence is not found in other aminotransferases (Mehta et al. 1989), and, therefore, its functional significance is unclear. As far as is currently known, there are no similarities in the catalytic or functional properties of mammalian AGT and cyanobacterial soluble hydrogenase. Nevertheless, they show considerably more sequence identity to each other than does either AGT to other aminotransferases or TES to other hydrogenases, indicating that, although AGT and TES might be functionally disparate, they are homologous proteins and that they may have evolved independently from other aminotransferases or hydrogenases. Whether mammalian AGTs and cyanobacterial soluble hydrogenases actually do share any functional/catalytic properties awaits investigation.

Microbial hydrogenases: Primary structure, classification, signatures and phylogeny

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H 2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H 2 -uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophus H16. The fifth is the H 2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the ‘nickel active center’ of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site. The variation of the sequences in these regions might determine the specific interaction between the external electron transfer domains of hydrogenases and their electron carriers. For example, the C-terminal cysteine-histidine rich region of the small subunits are distinguishable between those of H 2-uptake, H 2-producing, MV-reducing, F420-reducing and NAD-reducing NiFe(Se)-hydrogenases.

Microbial hydrogenases: primary structure, classification, signatures and phylogeny.

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrogen production by cyanobacteria

Cyanobacteria are capable of biophotolysis, the light-driven splitting of water into hydrogen and oxygen, in reactions which involve the enzymes nitrogenase and hydrogenase. Anabaena cylindrica possesses an integral membrane hydrogenase and a soluble hydrogenase, both of which are nickel-dependent. The release of nitrogenase-catalyzed hydrogen can be controlled chemically and also by manipulating nickel levels in the growth medium, suggesting that selection of uptake hydrogenase-deficient mutants is an important objective for maximizing biophotolysis. We have cloned and sequenced the hydrogenase gene for the soluble hydrogenase of A. cylindrica with a view to genetically manipulating the hydrogen metabolism of this and other cyanobacteria.

Hydrogenase mutants ofAlcaligenes eutrophusH16 show alterations in the electron transport system

Mutations in the genes coding for the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus strain H16 significantly affected the expression of respiratory chain components. In lithoautotrophically grown wild type cells electron flow mainly proceeded via the cytochrome c oxidases. Mutants defective in the membrane-bound hydrogenase contained a 2- to 3-fold higher cytochrome a content than the wild type and cytochrome c oxidase of the aa3-type was preferentially used by these cells for substrate oxidation. Mutants impaired in the soluble hydrogenase revealed slow growth on hydrogen, presumably due to inefficient reverse electron flow mechanisms which provide the cells with NADH for autotrophic CO2-fixation. In this class of mutants the two quinol oxidases of the o- and d-type in addition to the co-type oxidase were the predominant electron-transport branches.