The Involvement of Hydrogenases 1 and 2 in the Hydrogen-Dependent Nitrate Respiration of Escherichia coli

The study of Escherichia coli mutants synthesizing either hydrogenase 1 (HDK203) or hydrogenase 2 (HDK103) showed that the nitrate-dependent uptake of hydrogen by E. coli cells can be accomplished through the action of either of these hydrogenases. The capability of the cells for hydrogen-dependent nitrate respiration was found to depend on the growth conditions. E. coli cells grown anaerobically without nitrate in the presence of glucose were potentially capable of nitrate-dependent hydrogen consumption. The cells grown anaerobically in the presence of nitrate exhibited a much lower capability for nitrate-dependent hydrogen consumption. The inhibitory effect of nitrate on this capability of bacterial cells was either weak (the mutant HDK203) or almost absent (the mutant HDK103) when the cells were grown in the presence of peptone and hydrogen. Hydrogen stimulated the growth of the wild-type strain and the mutant HDK103 (but not the mutant HDK203) cultivated in the medium with nitrate and peptone. These data suggest that hydrogenase 2 is much more active in catalyzing nitrate-dependent hydrogen consumption than hydrogenase 1.     

固氮鱼腥藻(Anabaena azotica Ley HB 686)氢酶的存在形式和性质

Anabaena azotica HB 686氢酶有可溶性和膜结合两种存在状态,其中膜结合氢酶占总吸氢活性的67％,是吸氢酶的主要存在形式。在分离过程中,这两种氢酶在DEAE(DE—52)柱上的洗脱行为不同;此外,膜结合氨酶与氢的亲和力和吸氢能力均较强,对低温更敏感。这些差异可能与这两种氨酶的结构与生理功能不同有关。     

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni-Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.     

Intracellular localization of the hydrogenase in Desulfotomaculum orientis

Abstract Cell extracts of Desulfotomaculum orientis , grown with H₂ plus sulfate as sole energy source, revealed hydrogenase activities between 0.3 and 2 μmol H₂ per min and mg protein when methyl viologen was used as electron acceptor. With benzyl viologen, methylene blue, FAD or FMN, lower activities were found; NAD was not reduced. The hydrogenase activity was strongly inhibited by CuCl₂; however, copper inhibition was not observed with whole cells, indicating that the hydrogenase is located intracellularly. After high-speed centrifugation of cell-free extracts, varying proportions, between 11 and 90%, of the hydrogenase were detected in the soluble fraction, the rest being associated with the membrane fraction.     

Bidirectional measurement of hydrogenase activity by the pH-stat method

The protons produced by the catalytic activity of hydrogenase in H2 evolution from dithionite-reduced methyl viologen or through benzyl viologen reduction by H2 gas are automatically titrated by a pH-stat device. This approach allows the measurement of hydrogenase activity and ensures the constancy of pH during the reaction in absence of buffers. Kinetic assays and pH and temperature-dependence experiments with Desulfovibrio gigas hydrogenase performed by this method basically confirm the results obtained with customary manometric assay.     

Nickel and iron incorporation into soluble hydrogenase of Alcaligenes eutrophus

Archives of Microbiology - Qualitative and quantitative determination of proteins of the soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H16 was done by...     

Characterization of catalytic properties of hydrogenase isolated from the unicellular cyanobacterium Gloeocapsa alpicola CALU 743

The main catalytic properties of the Hox type hydrogenase isolated from the Gloeocapsa alpicola cells have been studied. The enzyme effectively catalyzes reactions of oxidation and evolution of H2 in the presence of methyl viologen (MV) and benzyl viologen (BV). The rates of these reactions in the interaction with the physiological electron donor/acceptor NADH/NAD+ are only 3-8% of the MV(BV)-dependent values. The enzyme interacts with NADP+ and NADPH, but is more specific to NAD+ and NADH. Purification of the hydrogenase was accompanied by destruction of its multimeric structure and the loss of ability to interact with pyridine nucleotides with retained activity of the hydrogenase component (HoxYH). To show the catalytic activity, the enzyme requires reductive activation, which occurs in the presence of H2, and NADH accelerates this process. The final hydrogenase activity depends on the redox potential of the activation medium (E(h)). At pH 7.0, the enzyme activity in the MV-dependent oxidation of H2 increased with a decrease in E(h) from -350 mV and reached the maximum at E(h) of about -390 mV. However, the rate of H2 oxidation in the presence of NAD+ in the E(h) range under study was virtually constant and equal to 7-8% of the maximal rate of H2 oxidation in the presence of MV.     

Efficient Hydrogen-Dependent Carbon Dioxide Reduction by Escherichia coli

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A cytosolic ferredoxin-independent hydrogenase possibly mediates hydrogen uptake in Trichomonas vaginalis

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紫云英根瘤菌109菌株氢酶诱导表达

根瘤菌自生培养物的吸收分子氢系统,不但可以在自养或异养的固体培养基上表达,而且也可以在异养的液相培养基中表达。其表达受碳水化合物、氧和分子氢浓度诸环境因子调节。诱导氢酶须用指数后期的培养物进行;所有试验过的可利用的碳水化合物均可阻抑氢酶表达;而20m mol/L的KNO_3和NH_4Cl对氢酶表达也有阻抑作用。正交组合试验指出紫云英根瘤菌的高吸氢活性是在40％N_2,14％H_2,4％O_2以及4.2％CO_2的气相条件下表达。Ar取代H_2后,无吸氢活性;氢酶表达后,移去H_2,则表达停止。氧浓度稍高阻抑氢酶表达;氯霉素的阻抑表达效应与氧阻抑类似。     

Cyanobacterial hydrogen production

With the global attention and research now being focussed on looking for an alternative to fossil fuel, hydrogen is the hope of future. Cyanobacteria are highly promising microorganisms for biological photohydrogen production. The review highlights the advancement in the biology of cyanobacterial hydrogen production in recent years. It discusses the enzymes involved in hydrogen production, viz. hydrogenases and nitrogenases, various strategies developed by cyanobacteria to limit nitrogenase inactivation by atmospheric and photosynthetic O₂, different biochemical and physicochemical parameters influencing the commercial cyanobacterial hydrogen production and the methods opted by different researchers for eliminating them to obtain maximum and sustained hydrogen production. Integrating the existing knowledge, techniques and expertise available, much future improvement and progress can be made in the field. This revised version was published online in November 2006 with corrections to the Cover Date.     

微生物产氢机理的研究进展

微生物可以利用工业废弃物产生氢气,其产氢机理可以分成两种:光合产氢和发酵产氢。前者利用光能,后者利用代谢过程中产生的电子,分解有机物产氢。氢酶是产氢过程中的关键酶,催化氢的氧化或质子的还原。氢酶主要有[NiFe]氢酶和[Fe]氢酶两种,具有不同的结构,但催化机理是相似的。本文主要综述产氢微生物的种类、微生物产氢代谢途径和关键酶催化机理,并展望微生物产氢研究的发展方向。     

Purification and properties of 1-phosphofructokinase from Escherichia coli

Abstract The thermophilic facultatively phototrophic green bacterium Chloroflexus aurantiacus strain Ok-70-fl was shown to possess sulfide-repressed hydrogenase activity. Biosynthesis of the enzyme was severely repressed by S²⁻ (5.7 mM) and stimulated specifically by Ni²⁺ and by molecular hydrogen. The hydrogenase was shown to be localized in the cytoplasmic membrane and could be solubilized from the latter by the detergent Triton X-100 in a state forming one enzymatically active band ( M _r 170 × 10³) in polyacrylamide gels. In the membraneous state, the hydrogenase had its maximal activity at 73°C and was active with methyl viologen, methylene blue, menadione and flavins, but not with NAD or NADP as electron acceptors. Solubilization of the enzyme with Triton X-100 resulted in a drastic increase in the FAD/FMN-linked activity.     

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (βδ) and an NADH:acceptor oxidoreductase (αγ)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed. Received: 14 May 1996 / Accepted: 7 November 1996     

SURVEY OF SELECTED SEA WEEDS FOR SIMULTANEOUS PHOTOPRODUCTION OF HYDROGEN AND OXYGEN1

Ten seaweed species were surveyed for simultaneous photoevolution of hydrogen and oxygen. In an attempt to induce hydrogenase activity (as measured by hydrogen photoproduction) the seaweeds were maintained under anaerobiosis in CO₂-free seawater for varying lengths of time. Although oxygen evolution was observed in every alga studied, hydrogen evolution was not observed. One conclusion of this research is that, in contrast to the microscopic algae, there is not a single example of a macroscopic alga for which the photoevolution of hydrogen has been observed, in spite of the fact that there are now at least nine macroscopic algal species known for which hydrogenase activity has been reported (either by dark hydrogen evolution or light-activated hydrogen uptake). These results are in conflict with the conventional view that algal hydrogenase can catalyze a multiplicity of reactions, one of which is the photoproduction of molecular hydrogen. Two possible explanations for the lack of hydrogen photoproduction in macroscopic algae are presented. It is postulated that electron acceptors other than carbon dioxide can take up reducing equivalents from Photosystem I to the measurable exclusion of hydrogen photoproduction. Alternatively, the hydrogenase system in macroscopic algae may be primarily a hydrogen-uptake system with respect to light-activated reactions. A simple kinetic argument based on recent measurements of the photosynthetic turnover times of simultaneous light-activated hydrogen and oxygen production is presented that supports the second explanation.     

Immunocytochemical localization of the soluble NAD-dependent hydrogenase in cells of Alcaligenes eutrophus

Abstract The localization of the soluble NAD-dependent hydrogenase in cells of Alcaligenes eutrophus PHB⁻4 was investigated using the protein A-gold technique as a post-embedding immunoelectron microscopic procedure. The enzyme was found throughout the cytoplasm of the cells. Autotrophic cells harvested in the logarithmic phase of growth exhibited a higher degree of labeling as compared to autotrophic cells from the stationary growth phase. Heterotrophic cells showed an almost identical labeling intensity in all growth phases. In a substrate-shift experiment (from fructose to glycerol, performed in the stationary growth phase), high amounts of newly synthesized enzyme could be observed two hours after the shift. This enzyme was located, as inclusion bodies, in the DNA region of the cells.     

微藻光生物水解制氢技术

氢气是未来人类社会可持续发展的理想能源。介绍微藻太阳能光生物水解制氢的研究现状,重点讨论微藻光水解制氢的生物学原理。重点讨论微藻光解水制氢的酶学机理、工艺过程以及当前的主要研究方向。通过比较微藻固氮酶制氢、可逆产氢酶直接光水解制氢、可逆产氢酶间接光水解制氢等技术路线的优缺点,指出利用微藻可逆产氢酶两步法间接光水解制氢最具发展潜力,可望为21世纪的“氢能经济社会”提供大量的氢源。该技术成功的关键在于相关的基因工程和代谢调控研究取得重大突破。     

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L

Four strains, coded as UPM1132, UPM1133^T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the ‘R. leguminosarum group’: R. laguerreae FB206^T, R. leguminosarum USDA 2370^T, R. anhuiense CCBAU 23252^T, R. sophoreae CCBAU 03386^T, R. acidisoli FH13^T and R. hidalgonense FH14^T, and 99.6% sequence similarity with R. esperanzae CNPSo 668^T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133^T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28 °C and growth was observed in the ranges 8-34 °C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G + C content was 60.8 mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133^T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133^T (=CECT 9542^T = LMG 30526^T).     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997