A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method.

A new assay method for hydrogenase [EC 1.12.2.1] based on the enzymic electrode reaction of H2-H+ equilibrium has been established. The method is based on the experimental fact that the short-circuit current of the electric cell composed of an electrode with hydrogenase and methylviologen as the mediator of H2-H+ equilibrium and a saturated calomel electrode as the counter electrode, is practically proportional to the amount of hydrogenase in the cell. The new method is referred to as the "enzymic electric cell method." This technique has applications not only to routine activity assay but also to the direct determination of the time course of enzyme denaturation, which has not previously been possible.     

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U x mg protein-1 at pH 7.0 and exhibited an apparent Km for the ferredoxin of 1 microM. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U x mg protein-1 at pH 7.0. The apparent Km for the reduced ferredoxin was 7.5 microM. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent Km for H2 was 5 microM. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H2/CO2-grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO2 during growth on acetate, and to ferredoxin-linked H2 uptake coupled to the reduction of CO2 to the redox state of CO during growth on H2/CO2 or methanol.     

Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen

The active site of [NiFe] hydrogenase from Desulfovibrio species is composed of a binuclear Ni-Fe complex bearing three diatomic nonprotein ligands to Fe and three bridges between the two metals, two of which are thiolate side chains of the protein moiety. The third bridging atom in the enzyme isolated from D. vulgaris Miyazaki F was suggested to be sulfur species, but was suggested to be oxygen species in D. gigas enzyme. When the hydrogenase from D. vulgaris Miyazaki F was incubated under the atmosphere of H2, H2S was liberated from the enzyme only in the presence of its electron carrier, cytochrome c3 or methylviologen. The amount of H2S liberation was little in the absence of electron carrier or essentially null when the enzyme was incubated under N2. The amount of H2S liberated was about 37% of the hydrogenase contained in the reaction vial in molar basis. These observations are in agreement with the recent observation that the third bridging site at the Ni-Fe active site is vacant in the reduced form of the enzyme revealed by X-ray crystallography.     

Distribution of transition metal ions in multiple forms of Methanosarcina hydrogenase

There were significant levels of in vitro hydrogenase activity in Methanosarcina strains. The multiple forms of hydrogenase were observed in cell free extracts of cells grown on methanol. Strains having poor growth on H₂ : CO₂ had four forms while strains having normal growth on all substrates contained two forms of hydrogenase. These multiple forms differ in their charges as well as in their composition of transition metal ions. The strain having normal growth showed higher incorporation of ⁶³Ni²⁺ and ⁶⁵Zn²⁺. Both hydrogenases, A and D, of strain P₃ had methylviologen and F₄₂₀-reducing activity and contained Zn²⁺ and Co²⁺ respectively. Hydrogenases A and D of strains P₁ and P₄ also had similar characteristics whereas hydrogenases B and C had only methylviologen reducing activity.     

Partial characterization of an electrophoretically labile hydrogenase activity of Escherichia coli K-12

A mutant of Escherichia coli K-12 is described that is specifically impaired in only one hydrogenase isoenzyme. By means of Tn5-mediated insertional mutagenesis, a class of mutants was isolated (class I) that had retained 20% of the overall hydrogenase activity. As determined by neutral polyacrylamide gel electrophoresis, the mutant contained normal amounts of the hydrogenase isoenzymes 1 and 2. Therefore, the hydrogenase activity affected seemed to be electrophoretically labile and was called hydrogenase L. The presence of such an activity was recently suggested in various papers and was called isoenzyme 3. Hydrogenase L might be identical or part of the latter isoenzyme. By DEAE ion-exchange chromatography it could be separated from hydrogenases 1 and 2. Hydrogenase activity in the parent strain HB101, determined manometrically with cell-free preparations and methylviologen as the electron acceptor, immediately showed maximal activity. However, class I mutants showed a lag phase which was dependent on the protein concentration utilized in the assay. This suggested that the fast initial activity of HB101 was due to hydrogenase L. The enzyme or enzyme complex showed an Mr around 300,000 and a pH optimum between 7 and 8. Strong indications about its physiological role were provided by the finding that in class I mutants H2 production by the formate-hydrogen lyase pathway was unimpaired, whereas fumarate-dependent H2 uptake was essentially zero. Complementation with F-prime factor F'116 but not with F'143 and coconjugation and cotransduction experiments localized the mutation (hydL) close to metC at approximately 64.8 min.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.     

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.     

Studies on the Induction of Reversible Hydrogenase from Cyanobacterium Anabaena variabilis and its Properties

The reversible hydrogenase in vegetative cells of A. variabilis cultured on NH4+ or N-free medium was induced by sparging with N2 for 24 hours under light. Both anaerobic condition and illumination appear to be necessary for the induction of hydrogenase in this algae. The properties of the hydrogenase in cell-free extract obtained from the cells grown on two nitrogen sources are similar: (1) Both the enzymes are able to evolve H2 in the presence of reduced methyl viotogen as electron donor, and to uptake H2 in the presence of benzyl viologen as electron acceptor. (2) The enzymes posses the thermal stability and are stable to O2. (3) The optimum pH required for H2 evolution activity of the enzymes is 7.0–7 5. (4) The Km of the enzymes obtained from NH4+ grown cells and N-free grown cells is 300 mmol/l and 295 mmol/l, respectively. So the high Km measured here suggests that the enzymes in both cases function physiologically as H2 evolution. (5) The activities of both enzymes are inhibited by CO but are not affected by C2H2. The induced H2 evolution activity of the reversible hydrogenase in cells grown on NH4+ reached 1530 nmol H2/mg dry wt, h, which was 3 to 5 times higher than from cells grown on N-free medium. Our experiment results indicate that the appearance of heterocysts of A. variabilis cultured on N-free medium affects the synthesis of reversible hydrogenase and the regulation of its activity.     

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.     

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.     

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

Occurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120

Two distinct types of hydrogenase occur in Anabaena 7120 and are distinguishable in whole filaments by the application of selective assay methods. A reversible hydrogenase occurs both in heterocysts and vegetative cells and can be selectively assayed by measuring H2 evolution from reduced methyl viologen. Activities in aerobically grown filaments were low but could be increased by 2 to 3 orders of magnitude by growing cells microaerobically. The presence of the reversible hydrogenase was independent of the N2-fixing properties of the organism, and activity did not respond to added H2 in the culture. Illumination was necessary during derepression of the reversible hydrogenase, and addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea increased the amount of enzyme that was synthesized. An uptake hydrogenase occurred only in heterocysts of aerobically grown filaments, but a small amount of activity also was present in the vegetative cells of filaments grown microaerobically with 20% H2. It was assayed selectively by measuring an oxyhydrogen reaction at atmospheric levels of O2. Additional uptake hydrogenase could be elicited by including H2 or by removing O2 from the sparging gas of a culture.     

Hydrogenase activity of Chlorella vulgaris cells

Cell suspensions of Chlorella vulgaris were found to possess the hydrogenase activity as was confirmed by their ability to absorb H2 in the presence of benzyl viologen, azocarmine and other hydrogen acceptors as well as to produce H2 from reduced methyl viologen. Incubation of the cells in the dark under anaerobic conditions in the atmosphere of H2, N2 or Ar stimulated the activity of hydrogenase and induced its de novo synthesis. Treatment of the cells adapted to anaerobiosis with dry ice or liquid nitrogen considerably increased their hydrogenase activity. The enzyme of the adapted cells was more resistant to the inactivation by O2 and temperature.     

Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus

The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase.     

Hydrogenase activity of Rhodopseudomonas capsulata growing on organic media

When Rhodopseudomonas capsulata B10 grows in media with different organic compounds, the hydrogenase activity estimated both by the evolution and uptake of H2 is lowest in cells taken from the middle of the exponential growth phase, and highest in cells from the beginning of the stationary phase. Cells grown in a medium containing malate have a higher hydrogenase activity than those cultivated in a medium with lactate or other compounds (900 and 20 nmoles of H2 per 1 min per 1 mg of protein, respectively). In the experiments with chloramphenicol (10(-5) M), organic compounds (not CO2) were shown to repress hydrogenase synthesis. When the cells were incubated in a medium without an organic substrate or in its presence, the exogenous H2 or H2 evolved as the result of nitrogenase action causes an increase in the activity of hydrogenase.     

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.     

Unconventional and effective methods for the regeneration of NAD(P) H in microorganisms or crude extracts of cells

Several yeasts, as well as aerobic and anaerobic bacteria catalyze the reduction of NAD and NADP in the presence of reduced methylviologen. The rates are usually much higher than those of reductions of unsaturated substrates by the organisms in cofermentations with carbohydrates. Since methylviologen can be continuously reduced at the cathode of an electrochemical cell it acts in catalytic amounts as a regenerable electron donor. Such systems may be superior to that with glucose as electron donor, because the NAD(P)H can be used exclusively for the reduction of the unsaturated substrate. The rate of the NAD(P)H formation depends very much on the organism and for the same organism on the growth procedure, the growth medium, the pretreatment of the cells, the pH, the buffer as well as on the ionic strength. Cells of Candida utilis which were frozen and thawed several times were superior to cells freshly harvested. Crude extracts revealed the best activities.Clostridia show the highest activities (up to 15 U per mg protein in the crude extract) and are suitable catalysts for the preparation of [4S-²H]NADH and [4S-²H]NADPH using ²H₂O-buffer in an electrochemical cell.The combinations of Alcaligenes eutrophus or Clostridium kluyveri and Candida utilis extracts in the presence of methylviologen are effective systems to reduce hydroxyacetone with hydrogen gas as electron donor or in an electrochemical cell. In this combination of microorganisms NADH is formed mainly by A. eutrophus or C. kluyveri and consumed for the reduction of hydroxyacetone by a reductase present in Candida utilis. The productivity numbers of such combinations are 10–30 times higher than those of yeasts alone.NAD(P)H regeneration, methylviologen-dependent NAD(P)H formation, deuterated NAD(P)H, Clostridia, yeast, bioreduction     

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.     

Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii

The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.