Heterologous functionality and roles of conserved cysteine motifs of the [NiFe]-hydrogenase accessory protein,HupK/HoxV

Numerous auxiliary proteins participate in the complex posttranslational modification process of [NiFe]-hydrogenases. In Thiocapsa roseopersicina, the HupK protein is important for the formation of active membrane-bound hydrogenases. The HupK proteins of various origins have moderate similarity to each other and to the large subunits of [NiFe]-hydrogenases. Site directed mutagenesis experiments were performed to disclose the role of the highly conserved cysteines in HupK. Cys54 was shown to be indispensable for the proper function of HupK and recreation of a large subunit like cysteine profile had negative effect on the activity of HupSL hydrogenase. Although, the results of the mutagenesis study slightly differed from that obtained for Ralstonia eutropha HoxV, it was clearly demonstrated that HupK from T. roseopersicina and HoxV from R. eutropha can substitute each other. It was also demonstrated that HoxV could be involved in the maturation of both Hup and Hyn hydrogenases in T. roseopersicina.     

Specificity and selectivity of HypC chaperonins and endopeptidases in the molecular assembly machinery of [NiFe] hydrogenases of Thiocapsa roseopersicina

The purple photosynthetic bacterium, Thiocapsa roseopersicina harbours at least three functional [NiFe] hydrogenases. Two of them are attached to the periplasmic membrane (HynSL, HupSL), while the third one is apparently localized in the cytoplasm (HoxEFUYH). Two hypC-type genes, coding for putative small maturation proteins, were found and their roles were studied by activity measurements performed with hypC mutants. Protein–protein interaction experiments confirmed that each HypC-type protein participates in the maturation of at least two [NiFe] hydrogenase large subunits via direct interaction. Endopeptidases perform the last step of the complex [NiFe] hydrogenase maturation process. A separate endopeptidase (HynD, HupD, HoxW) cleaves off the C-terminus of each large subunit and they are strictly specific for their corresponding hydrogenases. The results demonstrate a sophisticated assembly of these functionally active redox metalloenzymes through specific and selective protein–protein interactions and imply some diversity in the hydrogenase assembly machinery among the various microbes.     

Expression of the [FeFe] hydrogenase in the chloroplast of Chlamydomonas reinhardtii

Biological hydrogen generation from phototrophic organisms is a promising source of renewable fuel. The nuclear-expressed [FeFe] hydrogenase from Chlamydomonas reinhardtii has an extremely high turnover rate, and so has been a target of intense research. Here, we demonstrate that a codon-optimized native hydrogenase can be successfully expressed in the chloroplast. We also demonstrate a curiously strong negative selective pressure resulting from unregulated hydrogenase expression in this location, and discuss management of its expression with a vitamin-controlled gene repression system. To the best of our knowledge, this represents the first example of a nuclear-expressed, chloroplast-localized metalloprotein being synthesized in situ. Control of this process opens up several bioengineering possibilities for the production of biohydrogen.     

High-resolution crystallographic analysis of Desulfovibrio fructosovorans [NiFe] hydrogenase

Two 1.8 Å resolution crystal structures of an oxidised form of the [NiFe] hydrogenase of Desulfovibrio (D.) fructosovorans are reported. The high data quality allows for a detailed analysis of the active site geometry, confirming asymmetric bridging of the Ni and Fe ion by the two cysteine sulphur atoms and one oxygen atom as previously observed in the D. gigas enzyme. The CO ligand is now clearly distinguishable from the two CN⁻ ligands, as it refines to a significantly shorter distance to the Fe. The refined structures confirm the presence of long, mainly hydrophobic cavities that most probably provide pathways for H₂ diffusion between the molecular surface and the deeply buried active site. Amino acid sequence comparisons suggest that these cavities are significantly narrower in the so-called sensor hydrogenases, which may explain why this special class of enzymes is insensitive to O₂.     

Purification and characterization of a highly thermostable, oxygen-resistant, respiratory [NiFe]-hydrogenase from a marine, aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus

The membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 °C and 615 U/mg at 85 °C (the optimum temperature). The K_m and k_cat/K_m values for H₂ were, respectively, 12 μM and 8.58 × 10⁷ M⁻¹ s⁻¹. The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0-7.0. Results show that HmMBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 °C under H₂, and more than 72 h at 4 °C under air. The air-oxidized HmMBH for 72 h showed only weak EPR signals of Ni-B, suggesting a structural feature in which the active center is not easily oxidized.     

Maturation and processing of the recombinant [FeFe] hydrogenase from Desulfovibrio vulgaris Hildenborough (DvH) in Escherichia coli

The need of an efficient and well-characterized heterologous expression system of [FeFe]-hydrogenase for the production of O₂-resistant mutants prompted us to explore the use of Escherichia coli as a possible expression system. O₂-resistant hydrogenase mutants could be instrumental when coupling oxygenic photosynthesis with hydrogen bio-production. In general, expression of Desulfovibrio vulgaris Hildenborough active enzyme in E. coli was very modest indicating that the co-expression of the HydE, HydF and HydG maturases with hydrogenase structural genes in this bacterium is not optimal. A 28-fold increase in activity was obtained when these proteins were co-expressed with the Iron–Sulfur Cluster operon, indicating that one of the problems with over-expression is the correct insertion of FeS clusters. However, the measured activity is still about 4000-fold lower than the one measured in the native hydrogenase indicating that additional, so far unidentified factors may be necessary for optimal heterologous expression of [FeFe]-hydrogenase.     

Relationship between PHA and hydrogen metabolism in the purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS

Purple sulfur phototrophic bacteria accumulate various storage materials, such as sulfur globules, glycogen or polyhydroxy alkanoates (PHAs) under appropriate conditions. The formation of these materials requires reducing power which might be recovered upon their breakdown. This work aims at the understanding of the metabolism of PHA and its link to the nitrogenase mediated in vivo H₂ evolution in Thiocapsa roseopersicina BBS. The strain could accumulate 30% of the dry cell weight in the form of PHAs. Analysis of the genome sequence revealed the loci involved in PHAs synthesis and degradation. Phylogenetic analysis indicated independent evolution of the anabolic and catabolic proteins. A mutant carrying deleted PHA biosynthesis genes has been created in a host containing nitrogenase but none of the hydrogenases. Determination of the H₂ evolving capacity of the mutant revealed significantly reduced H₂ production in PHA deficient cells. Addition of excess electron sources such as thiosulfate stimulated the H₂ production via multiple effects.     

Conformational regulation of the hydrogenase gene expression in green alga Chlamydomonas reinhardtii

The direct relationship between hydrogenase gene conformation and its function in green alga Chlamydomonas reinhardtii has been investigated. We have analyzed the conformation in the 29 kilobase (kb) chromosome region containing [FeFe]-hydrogenase gene (hydA1) of C. reinhardtii in aerobic and anaerobic conditions using chromosome conformation capture technique (3C). The results showed a loop organization in the [FeFe]-hydrogenase gene region under aerobic conditions when the hydrogenase gene is silenced. In contrast, under anaerobic conditions, when the hydrogenase gene is active, no loop conformation in the gene region is present.     

Development of a PCR assay for the detection of nifH and nifD genes in indigenous photosynthetic bacteria

Molybdenum (Mo) nitrogenases consist of two components: dinitrogenase reductase (encoded by nifH) and the dinitrogenase or MoFe protein (encoded by nifDK). Nitrogenase enzyme of photosynthetic bacteria is responsible for hydrogen production. Therefore, primers were designed for the nitrogenase gene only. In this study, two primers (ND and NH) were designed after comparative genomic analysis of nifH and nifD gene sequences from public databases. The designed primers were used for the amplification of nifH and nifD genes to detect nitrogenase genes in photosynthetic bacteria. Initial detection was done using a monoplex Polymerase Chain Reactions (PCRs) followed by optimization of the PCR protocols. Subsequently, a duplex PCR was designed for amplification and detection of nifH and nifD genes in indigenous photosynthetic bacteria. Evaluation of the duplex PCR on six samples isolated from Palm Oil Mill Effluent (POME) showed that only four isolates contained both the nifH and nifD genes, indicating that these isolates were potential hydrogen-producing bacteria. PCR detection provides a rapid and efficient pre-identification of potential photosynthetic bacterial hydrogen producers.     

Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM^®-T-Easy cloning vector (pGEM^®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM^®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H₂/mol glucose, 1.68 fold higher than the wild type parent strain.     

Catalytic cycle of cytochrome-c3 hydrogenase,a [NiFe]-enzyme,deduced from the structures of the enzyme and the enzyme mimic

Hydrogenases catalyze uptake and production of H₂. Heterolytic cleavage of H₂ bound on [NiFe]-hydrogenase (E) produces two unequal H species to form E:H_aH_b, where H_a and H_b behave differently. The structures of various states of the enzyme established by crystallography and spectroscopy were used to construct a catalytic cycle of the enzyme. The Ni–Fe center of the active enzyme has the Ni–Fe bridging site vacant. The enzyme is suggested to bind H₂ either at Ni or Fe atom. In E:H_aH_b, H_a is considered to be a protein-bound hydron (proton or deuteron) at the entrance to the hydrophobic gas tunnel. The structure of a synthetic hydrogenase-mimic suggests H_b to be the 6th ligand to Fe. Two successive one-electron processes from E:H_aH_b complete the catalytic cycle of H₂ uptake. The reverse of the cycle operates in the H₂ production. The proposed catalytic cycle is consistent with the kinetic, crystallographic and spectroscopic studies.     

Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains

This study explored the genetic engineering of Escherichia coli for hydrogen (H₂) production. In E. coli W3110, the introduction of NAD⁺-reducing [NiFe]-hydrogenase from Cupriavidus necator, combined with the inactivation of three endogenous [NiFe]-hydrogenases, exhibited not only H₂ production but also H₂ uptake based on exogenous hydrogenase. Although the H₂ production ability was much lower than the H₂ uptake ability, inactivation of the ethanol, lactate, and succinate production pathways resulted in a marked increase in H₂ production, demonstrating the bidirectional hydrogenase function in vivo depending on NADH/NAD⁺. Unexpectedly, H₂ production was completely repressed under conditions for high expression of exogenous hydrogenase. Furthermore, the introduction of the heterologous enzyme markedly repressed the endogenous H₂ production ability of E. coli W3110 but not the HST02. These in vivo behaviors largely correlated with in vitro hydrogenase activity suggested complicated interactions between the native and nonnative functional expression of [NiFe]-hydrogenases.     

Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design

Phototrophic hydrogen production by indigenous purple non-sulfur bacteria, Rhodopseudomonas palustris PBUM001 from palm oil mill effluent (POME) was optimized using response surface methodology (RSM). The process parameters studied include inoculum sizes (% v/v), POME concentration (% v/v), light intensity (klux), agitation (rpm) and pH. The experimental data on cumulative hydrogen production and COD reduction were fitted into a quadratic polynomial model using response surface regression analysis. The path to optimal process conditions was determined by analyzing response surface three-dimensional surface plot and contour plot. Statistical analysis on experimental data collected following Box-Behnken design showed that 100% (v/v) POME concentration, 10% (v/v) inoculum size, light intensity at 4.0 klux, agitation rate at 250 rpm and pH of 6 were the best conditions. The maximum predicted cumulative hydrogen production and COD reduction obtained under these conditions was 1.05 ml H₂/ml POME and 31.71% respectively. Subsequent verification experiments at optimal process values gave the maximum yield of cumulative hydrogen at 0.66 ± 0.07 ml H₂/ml POME and COD reduction at 30.54 ± 9.85%.     

Continuous monitoring of the activation and activity of [NiFe]-hydrogenases by membrane-inlet mass spectrometry

The hydrogen–deuterium (H⁺/D₂) exchange reaction catalysed by [NiFe]-hydrogenases in the D₂/H₂O system has been used to study enzyme activation and activity by membrane-inlet mass spectrometry. The activation of the [NiFe]-hydrogenases from Thiocapsa roseopersicina (HynSL), Desulfovibrio fructosovorans (HynSL), Desulfomicrobium baculatum (HysSL), Rhodobacter capsulatus (HupUV), and of the bidirectional tetrameric HoxFUYH enzymes from Synechocystis PCC 6308 (Gloeocapsa alpicola) and Anabaena variabilis ATCC 29413 was determined in response to oxygen depletion and to reductant addition (molecular hydrogen, reduced methyl viologen). Natural physiological activators (NADH, NADPH) of the bidirectional [NiFe] hydrogenases could also be identified by the H⁺/D₂ exchange reaction. The data are discussed in the light of current models of hydrogenase catalytic mechanism.     

Development and application of a functional CE-SSCP fingerprinting method based on [Fe–Fe]-hydrogenase genes for monitoring hydrogen-producing Clostridium in mixed cultures

A Capillary Electrophoresis Single Strand Conformation Polymorphism (CE-SSCP) method based on functional [Fe–Fe]-hydrogenase genes was developed for monitoring the hydrogen (H₂)-producing clostridial population in mixed-culture bioprocesses. New non-degenerated primers were designed and then validated on their specific PCR detection of a broad range of clostridial hydA genes. The hydA-based CE-SSCP method gave a specific and discriminating profile for each of the Clostridium strains tested. This method was validated using H₂-producing mixed cultures incubated at temperatures ranging from 25 °C to 45 °C. The hydA CE-SSCP profiles clearly differed between temperatures tested. Hence, they varied according to variations of the H₂ production performances. The HydA sequences amplified with the new primer set indicated that diverse Clostridium strains impacted the H₂ production yields. The highest performances were related to the dominance of Clostridium sporogenes-like hydA sequences. This CE-SSCP tool offers highly reliable and throughput analysis of the functional diversity and structure of the hydA genes for better understanding of the H₂-producing clostridial population dynamics in H₂ dark fermentation bioreactors.     

Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides

Photosynthetic bacteria are favorable candidates for biological hydrogen production due to their high conversion efficiency and versatility in the substrates they can utilize. For large-scale hydrogen production, an integrated view of the overall metabolism is necessary in order to interpret results properly and facilitate experimental design. In this study, a summary of the hydrogen production metabolism of the photosynthetic purple non-sulfur (PNS) bacteria will be presented.Practically all hydrogen production by PNS bacteria occurs under a photoheterotrophic mode of metabolism. Yet results show that under certain conditions, alternative modes of metabolism—e.g. fermentation under light deficiency—are also possible and should be considered in experimental design.Two enzymes are especially critical for hydrogen production. Nitrogenase promotes hydrogen production and uptake hydrogenase consumes hydrogen.Though a wide variety of substrates can be used for growth, only a portion of these is suitable for hydrogen production. The efficiency of a certain substrate depends on factors such as the activity of the TCA cycle, the carbon-to-nitrogen ratio, the reduction-state of that material and the conversion potential of the substrate into alternative metabolites such as PHB.All these individual components of the hydrogen production interact and are subject to strict regulatory controls. An overall scheme for the hydrogen production metabolism is presented.     

Effect of inactivation of genes involved in ammonium regulation on the biohydrogen production of Rhodobacter capsulatus

Hydrogen production by nitrogenase is an energetically expensive process for the cell, hence strictly controlled at different levels. Ammonium is one of the substances regulating nitrogenase activity. The key proteins in the regulation of nitrogenase by ammonium are two regulatory proteins; GlnB and GlnK. In order to increase hydrogen production of Rhodobacter capsulatus DSM1710 (wild type strain) grown on agricultural materials/wastes, ammonium inhibition of nitrogenase enzyme has to be eliminated. In this study, GlnB and GlnK were targeted to be inactivated by in frame site-directed mutagenesis. The glnB mutant R. capsulatus (GP1 strain) was obtained at the end of mutagenesis studies. In the case of glnK, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained.The effect of ammonium on the growth and hydrogen production of R. capsulatus GP1 was investigated and compared with DSM1710. Both DSM1710 and GP1 strains were effectively utilized acetate. The mutation did not affect cell growth significantly at different ammonium levels. Ammonium negatively affected hydrogen production of GP1 strain as well as the DMS1710. However, hydrogen production was significantly low in GP1 strain. The ammonium inhibition of hydrogen production could not be removed in glnB mutant probably due to the presence of an active GlnK protein in the cell. Therefore, GlnK has much more important role in the ammonium dependent control of nitrogenase than GlnB does. The growth and hydrogen production kinetics of R. capsulatus DSM1710 and GP1 were modelled. They were shown to fit to Logistic Model and Modified Gompertz Model, respectively.     

Characterization and overexpression of a [FeFe]-hydrogenase gene of a novel hydrogen-producing bacterium Ethanoligenens harbinense

Ethanoligenens, a novel ethanologenic and hydrogen-producing genus, has capability of hydrogen production at low pH. A [FeFe]-hydrogenase gene with [4Fe-4S] and [2Fe-2S] clusters from Ethanoligenens harbinense YUAN-3 was cloned and overexpressed in a non-hydrogen-producing Escherichia coli BL-21. This hydA gene consisted of an open reading frame of 1743 bp encoding 580 amino acids with an estimated molecular weight of 63 188.1 Da. Six characteristic sequence signatures were present within the H-cluster domain of [FeFe]-H₂ases, and three of them were described previously. The overexpressed and purified hydrogenases from recombinant cells showed catalytic activity in vitro and in vivo.     

Molecular characterization and homologous overexpression of [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1

The H₂-evoving [FeFe]-hydrogenase in Clostridium tyrobutyricum JM1 was isolated to elucidate molecular characterization and modular structure of the hydrogenase. Then, homologous overexpression of the hydrogenase gene was for the first time performed to enhance hydrogen production. The hydA open reading frame (ORF) was 1734-bp, encodes 577 amino acids with a predicted molecular mass of 63,970 Da, and presents 80% and 75% identity at the amino acid level with the [FeFe]-hydrogenase genes of Clostridium kluyveri DSM 555 and Clostridium acetobutylicum ATCC 824, respectively. One histidine residue and 19 cysteine residues, known to fasten one [2Fe–2S] cluster, three [4Fe–4S] clusters and one H-cluster, were conserved in hydA of C. tyrobutyricum.