Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.     

Reduction of technetium(VII) by Desulfovibrio fructosovorans is mediated by the nickel-iron hydrogenase

Resting cells of the sulfate-reducing bacterium Desulfovibrio fructosovorans grown in the absence of sulfate had a very high Tc(VII)-reducing activity, which led to the formation of an insoluble black precipitate. The involvement of a periplasmic hydrogenase in Tc(VII) reduction was indicated (i) by the requirement for hydrogen as an electron donor, (ii) by the tolerance of this activity to oxygen, and (iii) by the inhibition of this activity by Cu(II). Moreover, a mutant carrying a deletion in the nickel-iron hydrogenase operon showed a dramatic decrease in the rate of Tc(VII) reduction. The restoration of Tc(VII) reduction by complementation of this mutation with nickel-iron hydrogenase genes demonstrated the specific involvement of the periplasmic nickel-iron hydrogenase in the mechanism in vivo. The Tc(VII)-reducing activity was also observed with cell extracts in the presence of hydrogen. Under these conditions, Tc(VII) was reduced enzymatically to soluble Tc(V) or precipitated to an insoluble black precipitate, depending on the chemical nature of the buffer used. The purified nickel-iron hydrogenase performed Tc(VII) reduction and precipitation at high rates. These series of genetic and biochemical approaches demonstrated that the periplasmic nickel-iron hydrogenase of sulfate-reducing bacteria functions as a Tc(VII) reductase. The role of cytochrome c(3) in the mechanism is also discussed.     

Selenium increases hydrogenase expression in autotrophically cultured Bradyrhizobium japonicum and is a constituent of the purified enzyme.

We have investigated the effect of added selenite on autotrophic growth and the time course of hydrogen oxidation derepression in Bradyrhizobium japonicum 122DES cultured in a medium purified to remove selenium compounds. In addition, hydrogenase was purified to near homogeneity and examined for the specific incorporation of Se into the enzyme. The addition of Se at 0.1 microM significantly increased total cell protein and hydrogenase specific activity of harvested cells. Also, the addition of SeO3(2-) enhanced the time course of hydrogenase derepression by 133%, whereas VO3, AsO2(2-), SO2(2-), and TeO3(2-) failed to substantially affect hydrogenase derepression. During the final chromatographic purification of hydrogenase, a striking coincidence in peaks of protein content, Se radioactivity, and hydrogenase activity of fractions was obtained. The total Se content expressed per milligram of protein increased manyfold during the purification procedure. The mean Se content of the purified hydrogenase was 0.56 +/- 0.13 mol of Se per mol of enzyme. These results indicate that Se is an important element in the H2 metabolism of B. japonicum and that hydrogenase from B. japonicum is a seleno protein.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities

In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.     

The HypB protein from Bradyrhizobium japonicum can store nickel and is required for the nickel-dependent transcriptional regulation of hydrogenase

The HypB protein from Bradyrhizobium japonicum is a metal-binding GTPase required for hydrogenase expression. In-frame mutagenesis of hypB resulted in strains that were partially or completely deficient in hydrogenase expression, depending on the degree of disruption of the gene. Complete deletion of the gene yielded a strain (JHΔEg) which lacked hydrogenase activity under all conditions tested, including the situation as bacteroids from soybean nodules. Mutant strain JHΔ23H lacking only the N-terminal histidine-rich region (38 amino acids deleted, 23 of which are His residues) expressed partial hydrogenase activity. The activity of strain JHΔ23H was low in comparison to the wild type in 10–50 nM nickel levels, but could be cured to nearly wild-type levels by including 50 μM nickel during the derepression incubation. Studies on strains harbouring the hup promoter–lacZ fusion plasmid showed that the complete deletion of hypB nearly abolished hup promoter activity, whereas the histidine deletion mutant had 60% of the wild-type promoter activity in 50 μM NiCl₂. Further evidence that HypB is required for hup promoter-binding activity was obtained from gel-shift assays. HypB could not be detected by immunoblotting when the cells were cultured heterotrophically, but when there was a switch to microaerobic conditions (1% partial pressure O₂, 10% partial pressure H₂) HypB was detected, and its expression preceded hydrogenase synthesis by 3–6 h. ⁶³Ni accumulation by whole cells showed that both of the mutant strains accumulate less nickel than the wild-type strain at all time points tested during the derepression incubation. Wild-type cultures that received nickel during the HypB expression-specific period and were then washed and derepressed for hydrogenase without nickel had activities comparable to those cells that were derepressed for hydrogenase with nickel for the entire time period. In contrast to the wild type, strain JHΔ23H cultures supplied with nickel only during the HypB expression period achieved hydrogenase activities that were 30% of those cultures supplied with nickel for the entire hydrogenase derepression period. These results indicate that the loss of the metal-binding area of HypB causes a decrease in the ability of the cells to sequester and store nickel for later use in one or more hydrogenase expression steps.     

hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX. Received: 15 June 1998 / Accepted: 5 August 1998     

HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum

A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions. Derepressing conditions that induce hydrogenase activity in free-living, wild-type B. japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant. The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation. Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region. Crude protein extracts isolated from a B. japonicum hoxA mutant do not contain this binding compound. The HoxA protein was overexpressed in E. coli and isolated in the form of a maltose-binding protein (MBP)–HoxA fusion. The MBP–HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.     

The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein.

The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli. At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R. Waugh and D. H. Boxer, Biochimie 68:157-166, 1986). To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product. HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM. The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1. The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM. A chromatography system was established to measure nickel insertion into hydrogenase 3 from E. coli and to determine the effects of lesions in hypB. Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel. The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.     

Regulation of hydrogenase activity in vegetative cells of Anabaena variabilis.

Heterocyst-free (NH4+-grown) cultures of the cyanobacterium Anabaena variabilis produce a hydrogenase which is reversibly inhibited by light and O2. White or red light at an intensity of 5,000 lx inhibited greater than 95% of the activity. Oxygen at concentrations as low as 0.5% inhibited more than 85% of the hydrogenase in the vegetative cells of CO2-NH4+-grown cultures. The vegatative cell hydrogenase is also sensitive to strong oxidants like ferricyanide. In the presence of strong reductants like S2O4(2-), hydrogenase activity was not inhibited by light. However, hydrogenase activity in the heterocysts was insensitive to both light (greater than 5,000 lx) and O2 (10%). Heterocysts and light-insensitive hydrogenase activity appear simultaneously during differentiation of the vegetative cells into heterocysts (an NH4+-grown culture transferred to NH4+-free, N2-containing medium). This light-insensitive hydrogenase activity was detected several hours before the induction of nitrogenase activity. These results suggest a mode of regulation of hydrogenase in the vegetative cells of A. variabilis that is similar to "redox control" of hydrogenase and other "anaerobic" proteins in enteric bacteria like Escherichia coli.     

Regulation of hydrogenase formation is temperature sensitive and plasmid coded in Alcaligenes eutrophus

Alcaligenes eutrophus grew well autotrophically with molecular hydrogen at 30 degrees C, but failed to grow at 37 degrees C (Hox Ts). At this temperature the strain grew well heterotrophically with a variety of organic compounds and with formate as an autotrophic substrate, restricting the thermolabile character to hydrogen metabolism. The soluble hydrogenase activity was stable at 37 degrees C. The catalytic properties of the wild-type enzyme were identical to those of a mutant able to grow lithoautotrophically at 37 degrees C (Hox Tr). Soluble hydrogenase was not rapidly degraded at elevated temperatures since the preformed enzyme remained stable for at least 5 h in resting cells or was diluted by growth, as shown in temperature shift experiments. Immunochemical studies revealed that the formation of the hydrogenase proteins was temperature sensitive. No cross-reactivity was detected above temperatures of 34 degrees C. The genetic information of Hox resides on a self-transmissible plasmid in A. eutrophus. Using Hox Tr mutants as donors of hydrogen-oxidizing ability resulted in Hox+ transconjugants which not only had recovered plasmid pHG1 and both hydrogenase activities but also were temperature resistant. This is evidence that the Hox Tr phenotype is coded by plasmid pHG1.     

Regulation by molecular oxygen and organic substrates of hydrogenase synthesis in Alcaligenes eutrophus.

Chemoautotrophic growth of Alcaligenes eutrophus 17707 is inhibited by 20% oxygen in the gas phase. Lowering the oxygen concentration to 4% results in chloramphenicol-sensitive derepression of soluble and membrane-bound hydrogenase activity (and of soluble hydrogenase antigen), showing that oxygen inhibition is due at least in part to repression of hydrogenase synthesis. Mutations resulting in derepression of hydrogenase activity (and antigen) under 25% oxygen (Ose-) mobilized with a self-transmissable plasmid which is already known to carry genes necessary for hydrogenase expression. Plasmid-borne mutations resulting in loss of soluble hydrogenase activity have no effect on the Ose phenotype, but chromosomal mutations resulting in reduction or loss of both hydrogenase activities cannot be made Ose-. The Ose- mutation does not alter the thermostability of either hydrogenase, and soluble hydrogenase in the mutant reacts with complete identity with that of the wild type, indicating that the Ose- phenotype does not result from structural alterations in either enzyme. Ose- mutants are also relieved of normal hydrogenase repression by organic substrates, which aggravates hydrogenase-mediated inhibition of heterotrophic growth by hydrogen. Regulation of hydrogenase in Ose- strains of A. eutrophus 17707 is nearly identical to that of wild-type A. eutrophus strains H1 and H16.     

Expression of hydrogenase activity in barley (Hordeum vulgare L.) after anaerobic stress

Germinated seeds of barley, after anaerobic treatment, produce considerable amounts of hydrogen. The anaerobic stress induces hydrogenase activity, as measured by the ability to evolve hydrogen from reduced methyl viologen. Several pieces of evidence, including bacteriological controls and induction in axenic calluses, indicate that the hydrogenase activity is induced by the plant itself and not by contaminant microorganisms. The hydrogenase is selectively induced in roots and to a lower extent in hypocotyls, but no hydrogenase activity can be detected in leaves.     

Regulation of hydrogenase in Rhizobium japonicum.

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.     

Regulation of hydrogenase activity in enterobacteria.

Proteus vulgaris, Escherichia coli, and Citrobacter freundii cells were devoid of hydrogenase activity when grown on complex medium or minimal medium plus glucose in the presence of saturating levels of dissolved oxygen. Anaerobically grown cells had appreciable hydrogenase activity. Cells grown anaerobically in the presence of CO (an inhibitor of hydrogenase) or nitrate (an electron acceptor) lacked hydrogenase activity. To make hydrogenase essential for anaerobic growth, cells were grown on fumarate, a nonfermentable carbon source. P. vulgaris and C. freundii evolved H2 gas under these conditions, and the hydrogenase-specific activity was 8 to 10 times greater than that in cells grown on glucose. Cell growth was inhibited by CO, and the cells grew but lacked hydrogenase activity when grown in the presence of nitrate. E. coli grew on fumarate plus H2, and the specific activity was five times greater than that in cells grown on glucose. Thus, hydrogenase activity is inducible and is expressed maximally when the enzyme is essential for cellular growth. Under conditions of growth where the enzyme would not be catalytically active, cells contain little active hydrogenase. Under anaerobic conditions where the enzyme is not essential for growth, the level of hydrogenase activity is intermediate.     

Effect of chelating agents on hydrogenase in Azotobacter chroococcum. Evidence that nickel is required for hydrogenase synthesis.

The chelating agents EDTA, o-phenanthroline, nitrilotriacetic acid (NTA), ethylenediamine-bis(o-hydroxyphenylacetic acid) (EDDA) or dimethylglyoxime prevented the expression of hydrogenase activity in batch cultures of nitrogen-fixing Azotobacter chroococcum, but did not inhibit preformed enzyme. The inhibition was reversed either by adding a mixture of trace elements (Cu2+, Mn2+, Zn2+, Co2+) or Ni2+ or, to a lesser degree, Co2+ alone. Ni2+ or Ni2+ + Fe2+ also enhanced the rate of hydrogenase derepression in A. chroococcum in the absence of any added chelator, if the medium was first extracted with 8-hydroxyquinoline. A. chroococcum accumulated 63Ni2+ by an energy-independent mechanism. Both, Ni2+ uptake and hydrogenase synthesis were equally inhibited by either NTA, EDTA, EDDA or dimethylglyoxime. The evidence suggests a role for Ni2+ in hydrogenase synthesis.     

Effects of electron mediator charge properties on the reaction kinetics of hydrogenase from Chlamydomonas

Anions modulate hydrogenase activity in cell-free preparations of Chlamydomonas reinhardtii, and this modulation is greatly influenced by the charge properties of the redox agent included to mediate electron transfer to hydrogenase. With cationic methyl viologen as the electron mediator, anions stimulate the maximum velocity of H2 production (e.g., a 320% increase in the presence of 1 M NaCl) but have little effect on the Km for methyl viologen. Conversely, when hydrogenase activity is mediated by polyanionic metatungstate or ferredoxin, H2 production is strongly inhibited by anions (e.g., 70-77% inhibition by 0.2 M NaCl). This inhibition is primarily due to a reduced affinity of hydrogenase for these mediators (as evidenced by a large increase in Km values), rather than a change in the maximum velocity of the reaction. Anions have little effect on the kinetics of hydrogenase activity mediated by zwitterionic sulfonatopropyl viologen, a redox agent with a nearly neutral net charge. These results suggest the presence of a cationic region near the active site of hydrogenase. This cationic region, probably due to lysine and/or arginine residues, may serve in vivo to facilitate the interaction between hydrogenase and ferredoxin, the polyanionic, physiological electron mediator.     

Immobilization of Desulphovibrio desulphuricans: cell-associated hydrogenase in beaded matrices

The immobilization of the hydrogenase (cytochrome c₃ hydrogenase, hydrogen: ferricytochrome c₃ oxidoreductase, EC 1.12.2.1) activity associated with Desulphovibrio desulphuricans whole cells is described. The periplasmic hydrogenase was prevented from leaking from the cells by glutaraldehyde treatment. This modification left the hydrogenase activity of the cells unchanged. The resulting whole-cell preparation was then immobilized in beaded gels formed by either calcium alginate or low temperature radiation-polymerized polyacrylamide(s). The alginate matrix was used to study the effect of bead diameter and retention of hydrogenase activity after immobilization. Polyacrylamide matrices were used to study the effects of immobilization on hydrogenase activity vs. pH profiles and also matrix charge effects on enzymatic activity and long-term storage.