Characterization of the oxygen tolerance of a hydrogenase linked to a carbon monoxide oxidation pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O2. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O2, 79% N2), its H2 evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O2 for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O2 inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 microM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O2, potential applications of this hydrogenase are discussed.     

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.     

Characterization of the hydrogen-deuterium exchange activities of the energy-transducing HupSL hydrogenase and H(2)-signaling HupUV hydrogenase in Rhodobacter capsulatus

Rhodobacter capsulatus synthesizes two homologous protein complexes capable of activating molecular H(2), a membrane-bound [NiFe] hydrogenase (HupSL) linked to the respiratory chain, and an H(2) sensor encoded by the hupUV genes. The activities of hydrogen-deuterium (H-D) exchange catalyzed by the hupSL-encoded and the hupUV-encoded enzymes in the presence of D(2) and H(2)O were studied comparatively. Whereas HupSL is in the membranes, HupUV activity was localized in the soluble cytoplasmic fraction. Since the hydrogenase gene cluster of R. capsulatus contains a gene homologous to hoxH, which encodes the large subunit of NAD-linked tetrameric soluble hydrogenases, the chromosomal hoxH gene was inactivated and hoxH mutants were used to demonstrate the H-D exchange activity of the cytoplasmic HupUV protein complex. The H-D exchange reaction catalyzed by HupSL hydrogenase was maximal at pH 4. 5 and inhibited by acetylene and oxygen, whereas the H-D exchange catalyzed by the HupUV protein complex was insensitive to acetylene and oxygen and did not vary significantly between pH 4 and pH 11. Based on these properties, the product of the accessory hypD gene was shown to be necessary for the synthesis of active HupUV enzyme. The kinetics of HD and H(2) formed in exchange with D(2) by HupUV point to a restricted access of protons and gasses to the active site. Measurement of concentration changes in D(2), HD, and H(2) by mass spectrometry showed that, besides the H-D exchange reaction, HupUV oxidized H(2) with benzyl viologen, produced H(2) with reduced methyl viologen, and demonstrated true hydrogenase activity. Therefore, not only with respect to its H(2) signaling function in the cell, but also to its catalytic properties, the HupUV enzyme represents a distinct class of hydrogenases.     

HupUV proteins of Rhodobacter capsulatus can bind H2: evidence from the H-D exchange reaction.

The H-D exchange reaction has been measured with the D2-H2O system, for Rhodobacter capsulatus JP91, which lacks the hupSL-encoded hydrogenase, and R. capsulatus BSE16, which lacks the HupUV proteins. The hupUV gene products, expressed from plasmid pAC206, are shown to catalyze an H-D exchange reaction distinguishable from the H-D exchange due to the membrane-bound, hupSL-encoded hydrogenase. In the presence of O2, the uptake hydrogenase of BSE16 cells catalyzed a rapid uptake and oxidation of H2, D2, and HD present in the system, and its activity (H-D exchange, H2 evolution in presence of reduced methyl viologen [MV+]) depended on the external pH, while the H-D exchange due to HupUV remained insensitive to external pH and O2. These data suggest that the HupSL dimer is periplasmically oriented, while the HupUV proteins are in the cytoplasmic compartment.     

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.     

Characterization of the Oxygen Tolerance of a Hydrogenase Linked to a Carbon Monoxide Oxidation Pathway in Rubrivivax gelatinosus

A hydrogenase linked to the carbon monoxide oxidation pathway in Rubrivivax gelatinosus displays tolerance to O₂. When either whole-cell or membrane-free partially purified hydrogenase was stirred in full air (21% O₂, 79% N₂), its H₂ evolution activity exhibited a half-life of 20 or 6 h, respectively, as determined by an anaerobic assay using reduced methyl viologen. When the partially purified hydrogenase was stirred in an atmosphere containing either 3.3 or 13% O₂ for 15 min and evaluated by a hydrogen-deuterium (H-D) exchange assay, nearly 80 or 60% of its isotopic exchange rate was retained, respectively. When this enzyme suspension was subsequently returned to an anaerobic atmosphere, more than 90% of the H-D exchange activity was recovered, reflecting the reversibility of this hydrogenase toward O₂ inactivation. Like most hydrogenases, the CO-linked hydrogenase was extremely sensitive to CO, with 50% inhibition occurring at 3.9 μM dissolved CO. Hydrogen production from the CO-linked hydrogenase was detected when ferredoxins of a prokaryotic source were the immediate electron mediator, provided they were photoreduced by spinach thylakoid membranes containing active water-splitting activity. Based on its appreciable tolerance to O₂, potential applications of this hydrogenase are discussed.     

Hydrogenase activity in Thiocapsa roseopersicina according to the D2--H20 metabolic reaction]

Extracts of Thiocapsa roseopersicina cells show hydrogenase activity, measured by evolution of H2 from reduced methylviologene (MV) and by D2-H2O exchange reaction. According to these reactions the most part of hydrogenases is found to be in the soluble fraction. Hydrogenase activity measured in the exchange reaction is completely inhibited by p-chloromercurybenzoate (5-10- minus 3 M), iodacetate (1-10- minus 2 M) and 26% inhibited by KCN and o-phenanthroline (5-10- minus 3 M). Evolution of H2 from reduced MV was not inhibited by o-phenanthroline, KCN and iodacetate and was inhibited by 66% only with p-chloromercurybenzoate. Light and ATP stimulated hydrogenase activity of chromatophores did not affect on its activity in the soluble fraction. The results obtained show that there are certain differences in hydrogenase systems responsible for the exchange reaction and evolution of H2.     

Hydrogenase activities in cyanobacteria

In the unicellular Anacystis nidulans, the expression of both the H2-uptake (with phenazine methosulfate or methylene blue as the electron acceptor) and H2-evolution (with methyl viologen reduced by Na2S2O4) was dependent on Ni in the culture medium. In extracts from Anacystis and Anabaena 7119, H2-evolution and uptake activities were strongly inhibited by Cu2+, p-chloromercuribenzoate and HgCl2 suggesting that at least one functional SH-group is involved in catalysis by hydrogenase. Extracts from the N2-fixing Anabaena 7119 contained two different hydrogenase fractions which could be separated by chromatography on DE-52 cellulose using a linear NaCl concentration gradient. The fraction eluting with 0.13 M NaCl from the column catalyzed only the uptake of H2 with methylene blue as the electron acceptor but virtually not the evolution of H2 ("uptake" hydrogenase fraction). The fraction eluting at a NaCl strength of 0.195 M catalyzed both H2-uptake with methylene blue and H2-evolution with reduced methyl viologen ("reversible" hydrogenase fraction). Growth under anaerobic conditions drastically enhanced the activity levels of the "reversible" but not of the "uptake" hydrogenase fraction. The "uptake" hydrogenase but not the "reversible" protein was activated by reduced thioredoxin. It is suggested that thioredoxin activates the H2-uptake by the membrane-bound "uptake" hydrogenase also in intact cells. The occurrence of the number of hydrogenases in cyanobacteria will be reevaluated.     

Nickel affects expression of the nickel-containing hydrogenase of Alcaligenes latus.

The effects of nickel on the expression of hydrogenase in the hydrogen-oxidizing bacterium Alcaligenes latus were studied. In the absence of added nickel, both hydrogenase activity, measured as O2-dependent H2 uptake, and hydrogenase protein, measured in a Western immunoblot, were very low compared with the levels in cells induced for hydrogenase in the presence of nickel. Hydrogenase activity and protein levels were dependent on the added nickel concentration and were saturated at 30 nM added Ni2+. The amount of hydrogenase protein in a culture at a given nickel concentration was calculated from the H2 uptake activity of the culture at that Ni2+ concentration. Between 0 and 30 nM added Ni2+, the amount of hydrogenase protein (in nanomoles) was stoichiometric with the amount of added Ni2+. Thus, all of the added Ni2+ could be accounted for in hydrogenase. Between 0 and 50 nM added Ni2+, all the Ni present in the cultures was associated with the cells after 12 h; above 50 nM added Ni2+, some Ni remained in the medium. No other divalent metal cations tested were able to substitute for Ni2+ in the formation of active hydrogenase. We suggest two possible mechanisms for the regulation of hydrogenase activity and protein levels by nickel.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus.

Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels.     

Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity. The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps. The enzyme was assayed by quantification of the H2:benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme. The enzyme constituted about 8% of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2. Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 mumol benzyl viologen reduced min-1 (mg protein)-1 (H2:benzyl viologen oxidoreductase). The final preparation contained polypeptides of apparent Mr 64,000, 31,000 and 29,000. Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates. The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the Mr-64,000 polypeptide. Immunological analysis revealed that the polypeptides of apparent Mr 31,000 and 29,000 are fragments of a single polypeptide of Mr 35,000 which is present in the detergent-dispersed membranes. The fragmentation of the Mr-35,000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme. A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35,000 polypeptide to one of 32,000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin. The enzyme in the detergent-dispersed membranes consists minimally of two subunits of Mr 64,000 and two subunits of Mr 35,000. It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200,000 g enzyme. The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+/- 0.20) mol Ni/200,000 g enzyme. A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme. The enzyme has a low Km for H2 (2.0 microM) in the H2:benzyl viologen oxidoreductase assay. It catalyses H2 evolution employing reduced methyl viologen as electron donor. It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.     

Properties of Stable Hydrogenase from the Purple Sulfur Bacterium Lamprobacter modestohalophilus

Some properties of a hydrogenase from the recently isolated phototrophic sulfur bacterium Lamprobacter modestohalophilus strain Syvash and its resistance to a number of inactivating factors have been investigated. The enzyme consists of two subunits, 64 and 30 kD; pI = 4.5. The optimal pH was 8.5-9.5 for hydrogen uptake and 4.0 for H2 evolution. Hydrogenase preparations were resistant to the effects of O2, CO, and temperature, revealing high stability under storage. A considerable inactivation of the enzyme was observed at temperatures above 80 degrees C; the temperature optimum of methyl viologen reduction by H2 was 85 degrees C. Inhibitory effects of Ni2+, Cd2+, and Mg2+ on the hydrogenase activity were shown to be reversible and competitive with respect to methyl viologen in the hydrogen oxidation reaction.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Monovalent nickel in hydrogenase from Chromatium vinosum. Light sensitivity and evidence for direct interaction with hydrogen

Redox titrations with hydrogenase from Chromatium vinosum show that its nickel ion can exist in 3, possibly 4, different redox states: the 3+, 2+, 1+ and possibly a zero valent state. The 1+ state is unstable: oxidation to Ni(II) occurs unless H2 gas is present. The Ni(I) coordination, but not that of Ni(III), is highly light sensitive. A photoreaction occurs on illumination. It is irreversible below 77 K, but reversible at 200 K. The rate of this photodissociation reaction in 2H2O is nearly 6-times slower than in H2O, indicating the breakage of a nickel-hydrogen bond. This forms the first evidence for an H atom in the direct coordination sphere of Ni in hydrogenase and for the involvement of this metal in the reaction with hydrogen.     

Hydrogen-induced structural changes at the nickel site of the regulatory [NiFe] hydrogenase from Ralstonia eutropha detected by X-ray absorption spectroscopy

For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RH(ox)) and presence of hydrogen (RH(+H2)). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH(+H2) state. XANES and EXAFS spectra indicate a coordination of the Ni in the RH(ox) and RH(+H2) that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H(2) are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH(ox)) to 8341.1 eV (RH(+H2)) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H(2). One likely interpretation of the data is the detachment of one sulfur ligand in RH(+H2) and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated Ni(II)(O,N)(2)S(3) for RH(ox) and six-coordinated Ni((III))(O,N)(3)X(1)S(2) [X being either an (O,N) or H ligand] for RH(+H2). Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed.     

Enhancement of endothelial cell migration by constitutively active LPA(1)-expressing tumor cells

2,3-Dihydroxybiphenyl-1,2-dioxygenase plays an important role in the degradation of polychlorinated biphenyls. The gene (BsbphCI) encoding a 2,3-DHBP dioxygenase from Bacillus sp. JF8 is 960 bp. We synthesized a 960 bp BsbphCI gene encoding a 2,3-DHBP dioxygenase derived from Bacillus sp. JF8 and expressed it in Escherichiacoli. The recombinant protein was about 36 kDa, confirmed by SDS-PAGE. The concentration of the purified protein was about 1.8 mg/mL. With 2,3-DHBP as a substrate, the optimal temperature for enzyme activity at pH 8.5 was 50 °C. The optimal pH for the 2,3-DHBP dioxygenase was 8.5. The enzyme retained 33% activity after heating at 60 °C for 60 min. We found that Cu(2+), K(+), Zn(2+), Mg(2+), Ni(2+), Co(2+), and Cd(2+) activated the enzyme. However, Ca(2+), Fe(2+), Li(+), and Cr(3+) inhibited it. Enzyme activity was reduced by exposure to H(2)O(2), SDS, and KI. The results of HPLC indicated that the transgenic E. coli strain with the BsbphCI gene degraded 2,3-DHBP more quickly than the wild type strain.     

Acquired tolerance to oxidative stress in Bifidobacterium longum 105-A via expression of a catalase gene

For improvement of tolerance to oxidative stress in Bifidobacterium longum 105-A, we introduced the Bacillus subtilis catalase gene (katE) into it. The transformant showed catalase activity (39 U/mg crude protein) in the intracellular fraction, which increased survival by ～100-fold after a 1-h exposure to 4.4 mM H(2)O(2), decreased de novo H(2)O(2) accumulation, and increased survival in aerated cultures by 10(5)-fold at 24 h. The protection level was better than that conferred by exogenously added catalase.