Photoproduction of H2 and NADPH2 by polyurethane-immobilized cyanobacteria

Summary Cyanobacterial cells were grown in polyurethane foams (polyester or polyvinyl types). Chlorogloea fritschii, Nostoc muscorum and Mastigocladus laminosus remained immobilized in the foams and were used for continuous photoproduction of H₂ from ascorbate with methyl viologen (MV) and hydrogenase or Pt catalysts, for periods in excess of 9 days. Foam-immobilized N. muscorum continuously photoreduced exogenous NADP for at least 24 h in the presence of Fd-NADP reductase with ascorbate as electron donor.     

Efficiency of hydrogen photoproduction by chloroplast-bacterial hydrogenase systems

A comparative study of H₂ photoproduction by chloroplasts and solubilized chlorophyll was performed in the presence of hydrogenase preparations of Clostridium butyricum. The photoproduction of H₂ by chloroplasts in the absence of exogenous electron donors, and with irreversibly oxidized dithiothreitol and cysteine, is thought to be limited by a cyclic transport of electrons wherein methylviologen short-circuits the electron transport in photosystem I. The efficiency of H₂ photoproduction by chloroplasts with ascorbate and NADPH is limited by a back reaction between light-reduced methylviologen and the oxidized electron donors. The use of a combination of electron donors (dithiothreitol and ascorbate), providing anaerobiosis without damage to chloroplasts, makes it possible to avoid consumption of reduced methylviologen for the reduction of oxidized electron donors and to exclude the short-circuiting of electron transfer. Under these conditions, photoproduction of H₂ was observed to occur with a rate of 350 to 400 micromoles H₂ per milligram chlorophyll per hour. In this case, the full electron-transferring capability of photosystem I (measured by irreversible photoreduction of methyl red or O₂) is used to produce H₂.     

Kinetic analyses of the OJIP chlorophyll fluorescence rise in thylakoid membranes

N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) was previously used to study the kinetics of the OJIP chlorophyll fluorescence rise. The present study is an attempt to elucidate the origin of TMPD-induced delay and quenching of the I–P step of fluorescence rise. For this purpose, we analyzed the kinetics of OJIP rise in thylakoid membranes in which electron transport was modified using ascorbate, methyl viologen (MV), and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). In the absence of TMPD, the OJIP kinetics of fluorescence induction (FI) was not altered by ascorbate. However, ascorbate eliminated the I–P rise delay caused by high concentrations of TMPD. On the other hand, neither ascorbate nor DBMIB, which blocks the electron release from Photosystem II (PS II) at the cytochrome b₆/f complex, could prevent the quenching of I–P rise by TMPD. In control thylakoids, MV suppressed the I–P rise of FI by about 60. This latter effect was completely removed if the electron donation to MV was blocked by DBMIB unless TMPD was present. When TMPD intercepted the linear electron flow from PS II, re-oxidation of TMPD by photosystem I (PS I) and reduction of MV fully abolished the I–P rise. The above is in agreement with the fact that TMPD can act as an electron acceptor for PS II. With MV, the active light-driven uptake of O₂ during re-oxidation of TMPD by PS I contributes towards an early decline in the I–P step of the OJIP fluorescence rise.     

Purification and properties of the H2-oxidizing (uptake) hydrogenase of the N2-fixing anaerobe Clostridium pasteurianum W5

Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H₂-oxidizing (uptake) hydrogenase. The H₂-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H₂ oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H₂ produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a M_r of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S⁼ contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H₂-production and H₂-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH₃-grown cells was much lower than that in N₂-fixing cells.     

Effect of anaerobiosis on photosynthetic reactions and nitrogen metabolism of algae with and without hydrogenase

Summary The effect of anaerobic (N₂+CO₂) pre-incubation in the dark on photosynthetic reactions (O₂ evolution, measured manometrically and with the oxygraph; fluorescence; and photoproduction of H₂, measured with the mass spectrometer) was studied in algae with hydrogenase (strains of Chlorella fusca, C. kessleri, C. vulgaris f. tertia, and Ankistrodesmus braunii) and in algae without hydrogenase (strains of Chlorella vulgaris, C. saccharophila, and C. minutissima).The inhibition by anaerobic incubation of photosynthetic O₂ evolution is much stronger in algae without hydrogenase than it is in algae with hydrogenase. The effect of anaerobiosis is most pronounced at rather low light intensity (about 1000 lux), in acid medium (pH 4), and after prolonged anaerobic incubation in the dark (about 20 h). These results indicate that the presence of hydrogenase might be ecologically advantageous for algae under certain conditions.Chlorophyll fluorescence showed the fastest response to anaerobic incubation, and the most pronounced difference between algae with and without hydrogenase. After only 30 min under N₂+CO₂, fluorescence in algae with hydrogenase starts with a peak and decreases within 10 to 20 sec to a rather low steady-state level which is only slightly higher than that found under aerobic conditions. In algae without hydrogenase, fluorescence is rather low during the first 1 to 2 sec and then rises to a higher steady-state level which is much higher than that of the aerobic controls. This indicates an inhibition due to anaerobiosis of photosystem II in algae without hydrogenase.Algae with hydrogenase can react in different ways during the first minutes of illumination. In some cases there is an immediate photoproduction of H₂, which is followed after a few minutes by photosynthetic O₂ evolution; in other algae there is a simultaneous production of H₂ and O₂ from the very beginning; in a few experiments there was no photoproduction of H₂ at all, and in this case there was no photosynthetic O₂ evolution either. Thus, photoproduction of H₂ seems to be the process which normally enables algae with hydrogenase to oxidise and thereby activate their photosynthetic electron transport system after anaerobic incubation.A mass spectrometric search for nitrogen fixation (using N₂ and acetylene) in eucaryotic green algae gave negative results, even with species containing hydrogenase and under anaerobic conditions.     

Efficiency of hydrogen photoproduction by photosystem I-enriched subchloroplast vesicles combined with Proteus mirabilis cells. Effects of some exogenous electron donors

High rates of hydrogen photoproduction are obtained when glutaraldehyde-fixed Photosystem I-enriched vesicles (Photosystem II-depleted) are added to hydrogenase-containing cells of Proteus mirabilis in the presence of the mediator methylviologen and a suitable electron donating system. This donor system includes ascorbate, dithioerythritol (DTE) and the mediator tetramethylphenylene-diamine (TMPD) and reduces the photosynthetic electron transfer chain at the level of plastocyanin. Both DTE and ascorbate are required for hydrogen photoproduction, DTE being the ultimate electron donor and ascorbate only having a catalytic function. Whereas the aerobic photoreduction of methylviologen is similar in the presence of DTE, ascorbate or both, under anaerobic conditions only combination of both compounds results in a high and stable amount of reduced methylviologen that can be utilized by the hydrogenase. It is concluded that oxidation reactions of reduced methylviologen, competing with the hydrogenase, rather than methylviologen photoreduction, limit hydrogen photoproduction in the presence of either DTE or ascorbate. These oxidation reactions are suggested to involve back reactions to the oxidized form(s) of ascorbate and DTE but backflow to the photosynthetic electron transfer chain (i.e. cyclic electron transfer) can not be excluded.Abbreviations Tes N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid - DTE 1,4-dithioerythritol - TMPD, N,N,N N-tetramethyl-p-phenylenediamine - DCMU 3-(3, 4-dichlorophenyl)-1, 1,-dimethylureum - EDAC 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide - DNP-INT 2-iodo-6-isopropyl-3-methyl-2, 4, 4-trinitrodiphenyl ether - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone - PS photosystem - Chl chlorophyll     

EDTA activation of H2 photoproduction by Rhodospirillum rubrum

Summary The effect of trace elements (Fe, Ni) and chelating compounds on the activity of hydrogen (H₂) uptake (Hup) hydrogenase, nitrogenase and rate and yield of H₂ photoproduction from l-lactate in photosynthetic cultures of Rhodospirillum rubrum was investigated. Hup activity depended on the availability of Ni²⁺ and was inhibited by EDTA (0.3–0.5 mm ethylenedinitrilotetraacetic acid). Addition of EDTA (0.5 mm) to the culture medium caused a nearly complete inactivation of Hup activity and activation of nitrogenase, which was paralleled by a threefold increase in total H₂ photoproduced from lactate. Hup^– mutants, isolated by transposon Tn5 mutagenesis, produced maximally twofold more H₂ than the wild-type. Experiments with different chelating agents [EDTA, NTA (nitrilotriacetic acid), citrate, isocitrate] and varying concentrations of Fe²⁺ and Fe³⁺ showed that photosynthetic growth and nitrogenase activity of R. rubrum were strongly influenced by the iron supply. It is concluded that EDTA enhanced H₂ photoproduction by (I) inhibition of biosynthesis of Hup hydrogenase and (II) mobilization of iron, thereby activating the biosynthesis of the nitrogenase complex. Correspondence to: M. Kern     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

The roles of weak light,oxygen and dithiothreitol in the photoreactivation of the oxygen evolving center of tris-washed and 2, 6-dichlorophenol indophenol-treated chloroplasts

The inactivated O₂-evolving center of Tris-washed chloroplasts was reactivated by DCPIP-treatment and photoreactivation in the presence of Mn²⁺, Ca²⁺, DTT and weak light. Many electron donors (Asc and reduced DCPIP, etc.) were found to be suitable substitutes for DTT. By studying the anaerobic inhibition of the reactivation, the electron acceptors O₂, NADP⁺, etc. were also found to be essential factors in photoreactivation. Weak light stimulated the chloroplast electron transport from the above-mentioned electron donors to the electron acceptor and effected the photoreactivation. More than 280 electrons were transported to NADP⁺ in the anaerobic photoreactivation of one unit of an O₂-evolving center with 400 Chl. Electron transport in the reactivation was inhibited by omitting DTT or Mn²⁺ ion, and by adding DCMU. The photoreactivated chloroplasts incorporated about 2 Mn by 400 Chl. Omission of DTT in the reactivation caused chloroplasts in the weak light to bind large amounts of excess Mn.Abbreviations Asc ascorbate - Chl chlorophyll - DCPIP 2, 6-dichlorophenol indophenol - DPC diphenyl carbazide - DTT dithiothreitol - Fd ferredoxin - STN a chloroplast preparation medium, containing 0.4 M sucrose, 0.05 M Tris-Cl and 0.01 M NaCl (pH 7.8 and 8.0) - TMPD tetramethyl-p-phenylenediamine     

Hydrogen oxidation activity in membranes from Rhizobium japonicum

Membranes capable of oxidizing H₂ with O₂ as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H₂-uptake specific activities when isolated in the presence of an H₂ atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O₂-dependent H₂-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H₂-uptake activity was sedimented. The particulate and soluble fractions containing H₂-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H₂ uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H₂ uptake with O₂ as acceptor in membranes was approx. 7.2. H₂-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H₂-reduced minus O₂-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H₂-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H₂-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H₂-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.     

Formation of the Photosynthetic Electron Transport System during the Early Phase of Greening in Barley Leaves

The development of photochemical activity in isolated plastids during the early phase of greening of 5-day-old etiolated barley seedlings was studied and related to the appearance of chlorophyll-protein complexes. Photochemical activities of PSI (DCIPH₂ → MV) and PSII (H₂O → DCIP, DPC → DCIP) appeared at 1 and 1.5 hours after the onset of illumination, respectively. However, PSI + PSII activity (H₂O → MV, H₂O → NADP) appeared at 4 hours. The functional plastoquinone pool was noticed, at the latest, from 4 hours. Chloroplast preparations from seedlings of 1 h of greening showed O₂ uptake upon illumination in the absence of MV (−MV activity). This activity peaked at 2 hours of greening, then fell to zero by 6 hours. In contrast to the −MV activity, MV-Hill activity began to increase at 2 hours. Although PSI activity appeared at 1 hour, it failed to reduce ferredoxin until 2 hours. NADP began to be photoreduced at 4 hours in accordance with the appearance of the ferredoxin:NADP reductase activity. After formation of PSI and PSII, electron transport systems between them and between PSI and NADP developed in coordination with each other. Thus, the whole electron transport from water to NADP began to operate at 4 hours.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Metabolically Engineered <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes

Three differently metabolically engineered strains, 2 single PHA^- and Hup^- mutants and one double PHA^-/Hup^- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H₂ photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H₂ recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup^- and the double PHA^-/Hup^- mutants, but not the single PHA^- mutant, exhibited increased rates of H₂ photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup^- mutant showed higher rates of H₂ production, but all 3 mutants sustained a longer-term H₂ photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H₂ evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H₂ photoproduction using actual waste-derived substrates.     

The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii

In nature, H₂ production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over‐reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress‐related genes, down‐regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H₂ production because it supplies electrons but the evolved O₂ inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50‐fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn‐cluster of PSII, and afterwards, it can donate electrons to tyrozin Z⁺ at a slow rate. This stage is followed by donor‐side‐induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H₂ production. We also show that ascorbate influenced H₂ evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.     

Increased Nitrogenase-Dependent H2 Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H₂ photoproduction. However, a mutation in a structural hup gene did not result in maximum H₂ production rates, indicating that the capacity to recycle H₂ was not completely lost. Highest H₂ production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H₂ recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Hydrogen production by photosystem I of Scenedesmus: Effect of heat and salicylaldoxime on electron transport and photophosphorylation

Summary Photosynthesis, photoreduction, the p-benzoquinone Hill reaction, and glucose uptake by whole cells, as well as cyclic photophosphorylation (with PMS) by chloroplast particles were strongly inhibited by 10^-2 M salicylaldoxime or by heating whole cells for 1–2 min at 55°. In contrast, H₂ photoproduction by whole cells of mutant No. 11 and wild type Scenedesmus and PS I-mediated MR reduction by chloroplast particles were either stimulated or not significantly inhibited by these agents. H₂ production by mutant No. 8 was slightly depressed by salicylaldoxime. DCMU inhibited H₂ photoproduction with 10^-2 M salicylaldoxime approximately 20%, indicating some contribution of electrons by endogenous organic compounds to photosystem II between the O₂-evolving mechanism and the DCMU-sensitive site. We conclude that photohydrogen production by PS I of Scenedesmus does not require cyclic photophosphorylation but is due to non-cyclic electron flow from organic substrate(s) through PS I to hydrogenase where molecular H₂ is released.The following abbreviations were used CI-CCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP dichlorophenol-indophenol - MR methyl red - PMS phenazine methosulfate - PS photosystem This work was supported by contract AT-(40-1)-2687 from the U.S. Atomic Energy Commission to Professor H. Gaffron.     

Utilization of free and immobilized Desulfovibrio hydrogenase in hydrogen photoproduction : Coupling efficiency of cytochrome C3, ferredoxin and flavodoxin

Summary Hydrogen photoproduction has been achieved by coupling free or immobilized hydrogenases from Desulfovibrio species to illuminated chloroplasts through different electron mediators. Whereas D. gigas flavodoxin or ferredoxin I cannot directly mediate the electron flux from chloroplasts to hydrogenase, the addition of these mediators considerably enhances the H₂ photoproduction of a system including cytochrome C₃. These immobilized hydrogenases exhibit good stability under working conditions and can be re-used.     

Purification and Characterization of Membrane-Associated Hydrogenase from the Deep-Sea Epsilonproteobacterium Hydrogenimonas thermophila

Membrane-associated hydrogenase was purified from the chemolithoautotrophic epsilonproteobacterium Hydrogenimonas thermophila at 152-fold purity. The hydrogenase was found to be localized in the periplasmic space, and was easily solubilized with 0.1% Triton X-100 treatment. Hydrogen oxidation activity was 1,365 μmol H₂/min/mg of protein at 80 °C at pH 9.0, with phenazine methosulphate as the electron acceptor. Hydrogen production activity was 900 μmol H₂/min/mg of protein at 80 °C and pH 6.0, with reduced methyl viologen as the electron donor. The hydrogenase from this organism showed higher oxygen tolerance than those from other microorganisms showing hydrogen oxidation activity. The structural genes of this hydrogenase, which contains N-terminal amino acid sequences from both small and large subunits of purified hydrogenase, were successfully elucidated. The hydrogenase from H. thermophila was found to be phylogenetically related with H₂ uptake hydrogenases from pathogenic Epsilonproteobacteria.     

Hydrogenase mediated nitrite reduction in chlorella

The assay of the hydrogenase of glucose-grown cells of Chlorella pyrenoidosa, strain 7-11-05 by means of nitrite reduction with molecular hydrogen is described. The hydrogenase of Chlorella shows maximum activity immediately after equilibration in the hydrogen atmosphere. The hydrogenase mediated reduction of nitrite to ammonia requires the presence of CO₂. However, at pH 6.4. when the reaction proceeds optimally, there is apparently sufficient retention of metabolic CO₂ to support the reaction, which goes to completion, at near maximum rates.

Reduction of nitrite in the hydrogenase system when CO₂ is present results in the uptake of 3 moles of H₂ per mole of nitrite and ammonia is the product. When CO₂ is absent or limiting, ammonia is also formed from nitrite but with the uptake of less than the stoichiometric amount of H₂. It is concluded that CO₂ is essential for the uptake of H₂, and that in the absence of CO₂ internal hydrogen donors support nitrite reduction.

The possibility that CO₂ exerts a catalytic effect in all reductions mediated by hydrogenase in algae is considered, and a further hypothesis, that hydrogenase arises from that portion of the photosynthetic machinery which also shows a catalytic requirement for CO₂, is proposed.