Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria.

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.     

The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems.

The heat of reaction (deltaH) of Fe(CN)63-, Methyl Viologen, FMN and FAD with S2O42- in aqueous buffer solutions was measured calorimetrically. In addition deltaH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0 values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.     

Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.     

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.     

Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe(3+) in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be -120mV. On reduction with Na(2)S(2)O(4) or Na(2)S(2)O(4)+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron-sulphur centre. The midpoint potential of this centre was very low, about -570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na(2)S(2)O(4), the iron-sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron-sulphur centre. Other ligands to haem, such as CO and the reaction product NH(3), had similar but less pronounced effects, and also changed the lineshape of the iron-sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na(2)S(2)O(4) in various proportions. Signals were interpreted as due to the reduced iron-sulphur centre (with slightly different g values), a haem-NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem-NO spectrum was more intense, whereas in the presence of an excess of Na(2)S(2)O(4) it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem-NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH(4) (+).     

Biodegradation of dibenzothiophene by thermophilic bacteria

Anaerobic microbial biodegradation of dibenzothiophene (DBT) was studied using thermophilic bacteria obtained from crude oil. A mixed culture was obtained that degraded 98% of DBT at 0.5 mg ml^–1 at 65 °C over 15 days both in the presence and in the absence of Methyl Viologen.     

Direct electroporation of clostridial hydrogenase into cyanobacterial cells

Clostridial hydrogenase was introduced into a cyanobacterium, Synechococcus elongatus, by direct electroporation. The introduced hydrogenase activity was 11 nmol H evolved with reduced Methyl Viologen per mg chlorophyll. The cells with clostridial hydrogenase showed simultaneous light-dependent evolution of H and O . This method will be applicable to assess a variety of hydrogenases for hydrogen production coupled with cyanobacterial photo-synthesis.     

Structural and catalytic properties of hydrogenase from Chromatium.

The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.     

Excellent wavelength-dependence of laser-induced delayed light-emission from mesophyll and bundle-sheath cells of maize.

Mesophyll and bundle-sheath cells from maize (Zea mays) were examined for delayed light-emission. An enzymic procedure was used to isolate the mesophyll and bundle-sheath cells. A tunable dye laser was used at 695-720 nm to excite delayed light-emission. The mesophyll cells show a typical Photosystem-II dependence on excitation wavelength for delayed light-emission, and this emission was eliminated by hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The bundle-sheath cells show no delayed light-emission with these excitation wavelengths. Photosystem-I emission was not observed from either cell type, there being no effect of Methyl Viologen on the observed signals.     

Control of magnesium–protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Role of light, oxygen, and electron and energy transfer

1. Magnesium-protoporphyrin chelatase activity, previously shown in whole cells of Rhodopseudomonas spheroides, could not be demonstrated in cell-free extracts prepared in different ways, although spheroplasts retained moderate activity. Slight activity was detected also in whole cells of Rhodospirillum rubrum. 2. The effects on the activity of the enzyme of inhibitors of electron and energy transfer were studied in whole cells of Rps. spheroides. Amytal, rotenone, azide and cyanide inhibited at low pO(2) in the dark but not under anaerobic conditions in the light. Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, as well as uncouplers and oligomycin, inhibited under all environmental conditions. 3. The effects on magnesium chelatase activity of intermediates of the tricarboxylic acid cycle, of thenoyltrifluoroacetone, of a number of artificial electron donors or acceptors, of various quinones and of the oxidation-reduction indicator dyes Benzyl Viologen and Methyl Viologen are described. 4. It was concluded that electron transport between a b-type and a c-type cytochrome as well as associated energy conservation and transformation reactions were essential for activity. There was also a specific requirement for ATP. 5. Exogenous protoporphyrin and magnesium protoporphyrin monomethyl ester were incorporated into bacteriochlorophyll or late precursors by whole cells. 6. Evidence is presented that the insertion of magnesium was the only step inhibited by oxygen in the biosynthetic pathway between protoporphyrin and bacteriochlorophyll.     

The reaction of Fe(III) myoglobin fluoride with reducing agents.

The reaction of reducing agents with Fe(III) myoglobin fluoride from sperm whale was studied at alkaline pH values. The rate of reduction by dithionite was indistinguishable from the rate of ligand dissociation even when the values of the rate constants for both were only 1.0 X 10(-3)S-1 (at pH 10.6). Reduction by the reduced Methyl Viologen radical ion and reduced Safranine was faster than the rate of dissociation, providing evidence that these reductants can donate electrons to the iron centre via a pathway involving an (undetectable) liganded Fe(II) intermediate.     

Altered Electron Flow in Continuous Cultures of Clostridium acetobutylicum Induced by Viologen Dyes

The physiological response of Clostridium acetobutylicum to methyl and benzyl viologen was investigated. Viologen dyes at low concentrations (at levels of parts per million [micrograms per milliliter]) caused significant metabolic shifts. Altered electron flow appeared to direct carbon flow from acid to alcohol production accompanied by decreased hydrogen evolution. Reducing equivalents normally released as free hydrogen were directed toward formation of NADH which, in turn, resulted in increased alcohol production. In addition, it was shown that solvent production can take place at pH 6.3. Contrary to previous reports, butanol production appears to be independent of high levels of acetate-butyrate and glucose.     

Photoinduced H2 production with Mg chlorophyll-a from Spirulina and colloidal platinum by visible light

Photoinduced H₂ production with Mg chlorophyll-a from Spirulina as a visible light photosensitizer by use of a three component system consisting of NADPH as an electron donor, Methyl Viologen as electron relay and colloidal platinum as catalyst was investigated. By using this system, the H₂ production rate was estimated to be 0.70 ± 0.03 × 10^–6 mol h^–1.     

Viologen dye inhibition of methane formation by Methanobacillus omelianskii

Wolin, E. A. (University of Illinois, Urbana), R. S. Wolfe, and M. J. Wolin. Viologen dye inhibition of methane formation by Methanobacillus omelianskii. J. Bacteriol. 87:993-998. 1964.-Low concentrations of methyl or benzyl viologen inhibit the formation of CH(4) from ethanol and CO(2) by washed cells of Methanobacillus omelianskii. Hydrogen, which is normally formed from ethanol, accumulates in greater quantities when CH(4) formation is inhibited by viologens. The viologens do not stimulate H(2) formation from ethanol in the absence of CO(2). Inhibition of CH(4) formation by the viologens is not reversed by H(2). A variety of other dyes and possible electron acceptors were tested for inhibition, and none was inhibitory in the same low-concentration range at which the viologens were effective.     

Purification of hydrogenases by affinity chromatography on Procion Red-agarose.

The agarose-coupled triazine dye Procion Red HE-3B has been demonstrated to be applicable as an affinity gel for the purification of five diverse hydrogenases, namely the soluble, NAD-specific and the membrane-bound hydrogenase of Alcaligenes eutrophus, the membrane-bound hydrogenase of the N2-fixing Alcaligenes latus, the reversible H2-evolving and the unidirectional H2-oxidizing hydrogenase of Clostridium pasteurianum. In the case of the soluble hydrogenase of A. eutrophus, chromatography on Procion Red-agarose even permitted the separation of inactive from active enzyme, thus yielding a 2-3-fold increase in specific activity. For the homogeneous enzyme preparation obtained after two column steps (Procion Red-agarose, DEAE-Sephacel), a specific activity of 121 mumol of H2 oxidized/min per mg of protein was determined. Kinetic studies with free Procion Red provided evidence that the diverse hydrogenases are competitively inhibited by the dye, each with respect to the electron carrier (NAD, Methylene Blue, Methyl Viologen), indicating a specific interaction between Procion Red and the catalytic centres of the enzymes. For the highly purified preparations of the soluble and the membrane-bound hydrogenase of A. eutrophus, in 50 mM-potassium phosphate, pH 7.0, Ki values for Procion Red of 103 and 19 microM have been determined.     

H2 production from maltose derived from starch using the visible light, photosensitization of Mg chlorophyll-a from Spirulina

A photoinduced H₂ production system, coupling d-maltose degradation by glucoamylase and glucose dehydrogenase (GDH) and H₂ production with colloidal platinum as a catalyst using the visible light-induced photosensitization of Mg chlorophyll-a (Mg Chl-a), has been developed. H₂ production was continuous when the reaction mixture containing d-maltose, glucoamylase, GDH, NAD⁺, Mg Chl-a, Methyl Viologen (MV²⁺, an electron relay reagent) and colloidal platinum was irradiated by visible light. The amount of H₂ production was estimated to be 5 ± 0.5 mol after 4 h irradiation. The yield of the conversion of d-maltose to H₂ in this system was ca. 1.8 %.     

The role of the membrane-bound hydrogenase in the energy-conserving oxidation of molecular hydrogen by Escherichia coli.

H2-dependent reduction of fumarate and nitrate by spheroplasts from Escherichia coli is coupled to the translocation of protons across the cytoplasmic membrane. The leads to H+/2e- stoicheiometry (g-ions of H+ translocated divided by mol of H2 added) is approx. 2 with fumarate and approx. 4 with nitrate as electron acceptor. This proton translocation is dependent on H2 and a terminal electron acceptor and is not observed in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide. H2-dependent reduction of menadione and ubiquinone-1 is coupled to a protonophore-sensitive, but 2-n-heptyl-4-hydroxy-quinoline N-oxide-insensitive, proton translocation with leads to H+/2e- stoicheiometry of approx. 2. H2-dependent reduction of Benzyl Viologen (BV++) to its radical (BV+) liberates protons at the periplasmic aspect of the cytoplasmic membrane according to the reaction: H2 + 2BV++ leads to 2H+ + 2BV+. It is concluded that the effective proton translocation observed in the H2-oxidizing segment of the anaerobic respiratory chain of Escherichia coli arises as a direct and inevitable consequence of transmembranous electron transfer between protolytic reactions that are spatially separated by a membrane of low proton-permeability.     

Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata.

Non-autotrophic ( Aut -) mutants of Rhodopseudomonas capsulata B10 were tested for their efficiency of nitrogenase-mediated H2 production. Three of these mutants ( IR3 , IR4 and IR5 ) showed an increase stoichiometry of H2 production, mediated by nitrogenase, from certain organic substrates. For example, in a medium containing 7 mM-L-glutamate as nitrogen source, strain IR4 produced 10-20% more H2 than did the wild type with DL-lactate or L-malate as major carbon source, 20-50% more H2 with DL-malate, and up to 70% more with D-malate. Strain IR4 was deficient in 'uptake' hydrogenase activity as measured by H2-dependent reduction of Methylene Blue or Benzyl Viologen. However, this observation did not explain the increased efficiency of H2 production, since H2 uptake (H2 recycling) was undetectable in cells of the wild type. Instead, increased H2 production by the mutant appeared to be due to an improved conversion of organic substrates to H2 and CO2, presumably due to an altered carbon metabolism. The metabolism of D-malate by different strains was studied. An NAD+-dependent D-malic enzyme was synthesized constitutively by the wild type, and showed a Km for D-malate of 3 mM. The activity of this enzyme was approx. 50% higher in strain IR4 than in the wild type, and the mutant also grew twice as fast as the wild type with D-malate as sole carbon source.     

Purification and properties of the membrane-bound by hydrogenase from Desulfovibrio desulfuricans.

The membrane-bound hydrogenase from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been purified to homogeneity, with an overall 80-fold purification and a specific activity of 70 mumol of H2 evolved/min per mg of protein. The hydrogenase had a relative molecular mass of 58 000 as determined by gel filtration and was estimated to contain six iron atoms and six acid-labile sulphur groups per molecule. The absorption spectrum of the enzyme was characteristic of an iron-sulphur protein. The E400 and E280 were 28 500 and 109 000 M-1.cm-1 respectively. The e.s.r. of the oxidized protein indicated the presence of [4Fe-4S]3+ or [3Fe-3S]3+, and another paramagnetic centre, probably Ni(III). The hydrogenase was inhibited by heavy-metal salts, carbon monoxide and high ionic strength. However, it was resistant to inhibition by thiol-blocking and metal-complexing reagents. N-Bromosuccinimide totally inhibited the enzyme activity at low concentrations. The enzyme was stable to O2 over long periods and to high temperatures. It catalyses both H2-evolution and H2-uptake with a variety of artificial electron carriers. D. desulfuricans cytochrome C3, its natural electron carrier, had a high affinity for the enzyme (Km = 2 microns). Rate enhancement was observed when cytochrome C3 was added to Methyl Viologen in the H2-evolution assay. The pH optimum for H2-evolution was 6.5.     

Purification and characterization of the ferredoxin component of 25-hydroxycholecalciferol 1 alpha-hydroxylase.

The chick kidney mitochondrial iron--sulphur protein (ferredoxin), a component of the NADPH--cytochrome P-450 reductase functional in the 1 alpha-hydroxylation of 25-hydroxycholecalciferol, was purified to homogeneity by chromatography on DEAE-cellulose, gel filtration on Sephadex G-100 and preparative electrophoresis on polyacrylamide gel. A novel NADPH--cytochrome c reductase assay utilizing crude renal NADPH--ferredoxin reductase was used for the detection of the ferredoxin. A mol. wt. of 53 000 was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by Sephadex G-100 gel filtration of the 125I-labelled ferredoxin. The ferredoxin has a sedimentation constant (S 20, w) of 2.66S, an A411/A280 of 0.4, and a molar absorptivity of 7300 cm-1 . M-1. The electron-paramagnetic-resonance spectrum after reduction with Methyl Viologen and dithionite was characteristic of ferredoxins with signals at g = 1.956 and 2.025. Two iron and two labile sulphur atoms per molecule of ferredoxin were released by acid. Ouchterlony immunodiffusion tests by using goat anti-(bovine adrenal ferredoxin) antiserum showed precipitin reactions with the bovine adrenal ferredoxin and the chick renal ferredoxin as antigens, suggesting that the renal ferredoxin shares antigenic determinants(s) with the natural adrenal antigen. Amino acid analysis showed that of the total number of residues per molecule of ferredoxin, glutamic acid and aspartic acid are the most abundant residues, comprising 17 and 15% respectively.