Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria.
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ABSTRACT:
SUBMITTER: Daday A
PROVIDER: S-EPMC1186349 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.
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Project description:Nitrogen fixation plays a crucial role in the nitrogen cycle by helping to convert nitrogen into a form usable by other organisms. Bacteria capable of fixing nitrogen are found in six phyla including Cyanobacteria. Molybdenum dependent nitrogenase (nif) genes are thought to share a single origin as they have homologs in various phyla. However, diazotrophic bacteria have a mosaic distribution within the cyanobacterial lineage. Therefore, the aim of this study was to determine the cause of this mosaic distribution. We identified nif gene operon structures in the genomes of 85 of the 179 cyanobacterial strains for which whole genome sequences were available. Four nif operons were conserved in each diazotroph Cyanobacterium, although there were some gene translocations and insertions. Phylogenetic inference of these genes did not reveal horizontal gene transfer from outside the phylum Cyanobacteria. These results support the hypothesis that the mosaic distribution of diazotrophic bacteria in the cyanobacterial lineage is the result of the independent loss of nif genes inherited from common cyanobacterial ancestors in each lineage.
| S-EPMC8071049 | biostudies-literature
Project description:BackgroundHydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above.ResultsWe identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28-41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions.ConclusionAnabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively. Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions.
| S-EPMC156652 | biostudies-literature
Project description:Plant endosymbiosis with nitrogen-fixing cyanobacteria has independently evolved in diverse plant lineages, offering a unique window to study the evolution and genetics of plant-microbe interaction. However, very few complete genomes exist for plant cyanobionts, and therefore little is known about their genomic and functional diversity. Here, we present four complete genomes of cyanobacteria isolated from bryophytes. Nanopore long-read sequencing allowed us to obtain circular contigs for all the main chromosomes and most of the plasmids. We found that despite having a low 16S rRNA sequence divergence, the four isolates exhibit considerable genome reorganizations and variation in gene content. Furthermore, three of the four isolates possess genes encoding vanadium (V)-nitrogenase (vnf), which is uncommon among diazotrophs and has not been previously reported in plant cyanobionts. In two cases, the vnf genes were found on plasmids, implying possible plasmid-mediated horizontal gene transfers. Comparative genomic analysis of vnf-containing cyanobacteria further identified a conserved gene cluster. Many genes in this cluster have not been functionally characterized and would be promising candidates for future studies to elucidate V-nitrogenase function and regulation.
| S-EPMC6645180 | biostudies-literature
Project description:BackgroundMolecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways.ResultsIn this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H2 production. The growth, H2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase.ConclusionsThis work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.
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Project description:The substrate-reducing proteins of all nitrogenases (MoFe, VFe, and FeFe) are organized as α2ß2(γ2) multimers with two functional halves. While their dimeric organization could afford improved structural stability of nitrogenases in vivo, previous research has proposed both negative and positive cooperativity contributions with respect to enzymatic activity. Here, a 1.4 kDa peptide was covalently introduced in the proximity of the P cluster, corresponding to the Fe protein docking position. The Strep-tag carried by the added peptide simultaneously sterically inhibits electron delivery to the MoFe protein and allows the isolation of partially inhibited MoFe proteins (where the half-inhibited MoFe protein was targeted). We confirm that the partially functional MoFe protein retains its ability to reduce N2 to NH3, with no significant difference in selectivity over obligatory/parasitic H2 formation. Our experiment concludes that wild-type nitrogenase exhibits negative cooperativity during the steady state regarding H2 and NH3 formation (under Ar or N2), with one-half of the MoFe protein inhibiting turnover in the second half. This emphasizes the presence and importance of long-range (>95 Å) protein-protein communication in biological N2 fixation in Azotobacter vinelandii.
| S-EPMC10207099 | biostudies-literature
Project description:Hydrogen with high energy content is considered to be a promising alternative clean energy source. Biohydrogen production through microbes provides a renewable and immense hydrogen supply by utilizing raw materials such as inexhaustible natural sunlight, water, and even organic waste, which is supposed to solve the two problems of "energy supply and environment protection" at the same time. Hydrogenases and nitrogenases are two classes of key enzymes involved in biohydrogen production and can be applied under different biological conditions. Both the research on enzymatic catalytic mechanisms and the innovations of enzymatic techniques are important and necessary for the application of biohydrogen production. In this review, we introduce the enzymatic structures related to biohydrogen production, summarize recent enzymatic and genetic engineering works to enhance hydrogen production, and describe the chemical efforts of novel synthetic artificial enzymes inspired by the two biocatalysts. Continual studies on the two types of enzymes in the future will further improve the efficiency of biohydrogen production and contribute to the economic feasibility of biohydrogen as an energy source.
| S-EPMC9919214 | biostudies-literature
Project description:Inspired by the active center of the natural [FeFe] hydrogenases, we designed a compact and precious metal-free photosensitizer-catalyst dyad (PS-CAT) for photocatalytic hydrogen evolution under visible light irradiation. PS-CAT represents a prototype dyad comprising π-conjugated oligothiophenes as light absorbers. PS-CAT and its interaction with the sacrificial donor 1,3-dimethyl-2-phenylbenzimidazoline were studied by steady-state and time-resolved spectroscopy coupled with electrochemical techniques and visible light-driven photocatalytic investigations. Operando EPR spectroscopy revealed the formation of an active [FeI Fe0 ] species-in accordance with theoretical calculations-presumably driving photocatalysis effectively (TON≈210).
| S-EPMC9313588 | biostudies-literature
Project description:It is now widely accepted that enzyme-catalysed C-H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data-especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs-are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.
| S-EPMC1647315 | biostudies-literature
Project description:When the MoFe (Kp1) and Fe (Kp2) component proteins of Klebsiella pneumoniae nitrogenase are incubated with MgADP and AlF4(-) in the presence of dithionite as a reducing agent, a stable putative transition-state complex is produced [Yousafzai and Eady (1997) Biochem. J. 326, 637-640]. Surprisingly, the EPR signal associated with reduced Kp2 is not detectable, but Kp1 retains the S=3/2 EPR signal arising from the dithionite reduced state of the MoFe cofactor centre of the protein. This is consistent with the [Fe4S4] centre of the Fe protein in the complex being oxidized, and similar observations have been made with the complex of Azotobacter vinelandii [Spee, Arendsen, Wassink, Marritt, Hagen and Haaker (1998) FEBS Lett. 432, 55-58]. No satisfactory explanation for the fate of the electrons lost by Kp2 has been forthcoming. However, we report here that during the preparation of the MgADP-AlF4 K. pneumoniae complex under argon, H2 was evolved in amounts corresponding to one half of the FeMoco content of the Kp1 (FeMoco is the likely catalytic site of nitrogenase with a composition Mo:Fe7:S9:homocitrate). This is surprising, since activity is observed during incubation in the absence of MgATP, normally regarded as being essential for nitrogenase function, and in the presence of MgADP, a strong competitive inhibitor of nitrogenase. The formation of H2 by nitrogenase in the absence of AlF4(-) was also observed in reaction mixtures containing MgADP but not MgATP. The reaction showed saturation kinetics when Kp1 was titrated with increasing amounts of Kp2 and, at saturation, the amount of H2 formed was stoichiometric with the FeMoco content of Kp1. The dependence of the rate of formation of H2 on [MgADP] was inconsistent with the activity arising from MgATP contamination. We conclude that MgATP is not obligatory for H+ reduction by nitrogenase since MgADP supports a very low rate of hydrogen evolution.
| S-EPMC1220184 | biostudies-other