Azotobacter Vinelandii Hydrogenase Research Articles

Comparison of isotope exchange, H 2 evolution, and H 2 oxidation activities of Azotobacter vinelandii hydrogenase

Azotobacter vinelandii hydrogenase was purified aerobically with a 35% yield. The purified enzyme catalyzed H 2 oxidation at much greater velocity than H 2 evolution. There was a large difference in activation energy for the two reactions. E A was 10 kcal/mol for H 2 oxidation and 22 kcal/mol for evolution. This difference in activation energies between the two reactions means that the ratio of oxidation velocity to evolution velocity drops from 70 at 33°C to 8 at 488°C. With D 2 and H 2O as substrates, both membranes and purified enzyme produced only H 2 and no HD in the isotope exchange reaction. The velocity of isotope exchange was equal to the velocity of H 2 evolution from reduced methyl viologen, indicating that the two reactions share the same rate-limiting step. D 2 and H 2 inhibited H 2 evolution, but D 2 did not inhibit isotope exchange. We conclude that H 2 and D 2 do not inhibit H 2 evolution by competing with H + for the active site of the reduced enzyme. The K m for D 2 in isotope exchange is 40-times greater than its K m in D 2 oxidation. The difference in K m cannot be accounted for by differences in k cat. We propose that redox environment regulates hydrogenase's affinity for D 2 (and likely H 2 as well).

Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution.

Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2.

In Azotobacter vinelandii hydrogenase, substitution of serine for the cysteine residues at positions 62, 65, 294, and 297 in the small (HoxK) subunit affects H2 oxidation [corrected

The essential role of the small (HoxK) subunit of hydrogenase of Azotobacter vinelandii in H2 oxidation was established. This was achieved by modification of the two Cys-X2-Cys amino acid motifs at the N and C termini of the HoxK subunit (Cys-62, -65, -294, and -297). The Cys codons were individually mutated to Ser codons. Modifications in these two motifs resulted in loss of hydrogenase activity. At the N terminus, the mutations of the codons for the motif Cys-62-Thr-Cys-64-Cys-65 decreased the activity of hydrogenase to levels no higher than 30% of those of the parental strain. H2 oxidation with the alternate electron acceptors methylene blue and benzyl viologen was decreased. H2 evolution and exchange activities were also affected. Cys-64 possibly substitutes for either Cys-62 or Cys-65, allowing for partial activity. Mutation of the codons for Cys-294 and Cys-297 to Ser codons resulted in no hydrogenase activity. The results are consistent with alterations of the ligands of FeS clusters in the HoxK subunit of hydrogenase [corrected].

Nucleotide sequence and organization of an H 2-uptake gene cluster from Rhizobium leguminosarum bv. viciae containing a rubredoxin-like gene and four additional open reading frames

The nucleotide sequence of a 3·2 kb region following the hydrogenase structural operon ( hupSLCDEF) in the H 2-uptake gene cluster from Rhizobium leguminosarum bv viciae strain 128C53 has been determined. Five closely linked genes encoding products of 16·3 (HupG), 30·5 (HupH), 8·0 (HupI), 18·4 (HupJ) and 38·7 (HupK) kDa were identified 166 bp downstream from hupF. Transposon insertions into hupG, hupH, hupJ and hupK suppress the H 2-oxidizing capability of the wild-type strain. The amino acid sequence deduced from hupI contains two Cys-X-X-Cys motifs, characteristic of rubredoxins, separated by 29 amino acid residues showing strong sequence homology with other bacterial rubredoxins. The amino acid-derived sequence from hupG and hupH showed homology to products from genes hyaE and hyaF of the operon encoding hydrogenase 1 from Escherichia coli, and hupJ and hupK were related to open reading frames identified in Rhodobacter capsulatus and Azotobacter vinelandii hydrogenase gene clusters. An involvement of the hupGHTJK gene cluster in redox reactions related to hydrogenase synthesis or activity is predicted on the basis of the function as electron carrier attributed to rubredoxin.

The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase.

The roles of the product of the hoxZ gene immediately downstream of the hydrogenase gene (hoxKG) in Azotobacter vinelandii were investigated by constructing and characterizing a mutant with the center of the hoxZ gene deleted. The strain lacking the functional hoxZ gene product exhibited a low rate of H2 oxidation with O2 as the electron acceptor relative to that of the wild-type strain. Nevertheless, when the enzyme was exogenously activated and methylene blue was used as the electron acceptor from hydrogenase, rates of H2 oxidation comparable to those in the wild-type strain were observed. These results suggest that the gene product of hoxZ plays a role in activating and maintaining hydrogenase in a reduced active state. The product of hoxZ could also be the linkage necessary for transfer of electrons from H2 to the electron transport chain.

Acetylene inhibition of Azotobacter vinelandii hydrogenase: acetylene binds tightly to the large subunit.

Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C2H2 proceeds more rapidly than the recovery of activity, which indicates that release of C2H2 is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with 14C2H2. Acetylene, or a derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidenced by SDS-PAGE and fluorography of 14C2H2-inhibited hydrogenase. This result suggests that C2H2, and by analogy H2, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from 14C2H2-inhibited protein during recovery. The inhibition is remarkably specific for C2H2: propyne, butyne, and ethylene are not inhibitors.

Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii

The effects of O{sub 2} on membrane-bound Azotobacter vinelandii hydrogenase in both the membrane-associated and purified states were characterized. O{sub 2} was a rapid-equilibrium, reversible inhibitor both of H{sub 2} oxidation/electron acceptor reduction activity and of the exchange activity. O{sub 2} inhibition was noncompetitive versus the electron acceptor methylene blue and uncompetitive versus the substrate H{sub 2}. O{sub 2} inhibition was rapidly reversed by removal of the O{sub 2}. In addition to an O{sub 2}-independent, reversible inhibition of hydrogenase, a time-dependent, irreversible inactivation by O{sub 2} of both H{sub 2} oxidation and exchange activities for this hydrogenase was demonstrated. The irreversible inactivation followed a first-order process. The inactivation by O{sub 2} was protected against by the substrate H{sub 2} in a concentration-dependent manner. This is the first report of H{sub 2} protection from O{sub 2}-dependent, irreversible inactivation for any hydrogenase. The competitive inhibitor CO did not affect the irreversible inactivation by O{sub 2} or the protection by H{sub 2}. A model is proposed that outlines various oxidation states of this hydrogenase and the effects of O{sub 2} on each state.

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition.

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.

Effects of alcohols on the reactivity and stability of Azotobacter vinelandii hydrogenase

The effects of alcohols on the reactivity of Azotobacter vinelandii hydrogenase were investigated. Hydrogenase catalyzed H 2 oxidation coupled to methylene blue, benzyl viologen, or phenazine methosulfate when in the presence of solvents containing 15 or 40% ethanol or 40% methanol or 2-propanol. In general, the K m 's for the electron acceptors were increased substantially by the presence of the alcohols, while the K m for H 2 was not altered in a solvent containing 40% ethanol. Calculation of the apparent maximum velocities for H 2 oxidation in the presence of alcohols indicated that the maximum velocity was not decreased in most cases. In contrast, the rates of both H 2 evolution and isotope exchange by hydrogenase were substantially decreased when in a solvent containing alcohol. Hydrogenase was inactivated in 100% ethanol with a half-life of 17 s. Hydrogenase from A. vinelandii was stable when stored in alcohol/buffer solvents at 20 °C or below. However, the thermal stability of hydrogenase was greatly decreased by inclusion of an alcohol in the solvent. When incubated at 55 °C in a solvent containing 40% ethanol, activity decreased in a first-order process with a half-life of 7 min. When incubated at the same temperature in aqueous buffer, no loss of activity was observed over 30 min.

Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase

The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.

Immunological and molecular evidence for a membrane-bound, dimeric hydrogenase in Rhodopseudomonas capsulata

Hydrogenase from Rhodopseudomonas capsulata membranes was purified to a specific activity of 37 μmol H 2 oxidized·min −1·mg −1. Analysis of the purified hydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed major protein-staining bands at 67 and 31 kDa that comigrated with the large and small subunits of Azotobacter vinelandii hydrogenase. Western immunoblots and enzyme-linked immunosorbent assays in microtiter plates demonstrated that the 67 and 31 kDa proteins in the R. capsulata hydrogenase are immunologically related to the large and small subunits, respectively, of the dimeric hydrogenases purified from A. vinelandii, Rhizobium japonicum, Alcaligenes latus, and Alcaligenes eutrophus H16 (membrane-bound). It is concluded that the photosynthetic microorganism, R. capsulata, contains a membrane-bound, dimeric hydrogenase that is immunologically related to the membrane-bound hydrogenase from the aerobic microorganisms mentioned.

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.