Nickel Affects Activity More Than Expression of Hydrogenase Protein in <Emphasis Type="Italic">Frankia</Emphasis>

The effects of nickel on hydrogen uptake and the post-translational processing of the large subunit of the hydrogenase protein in three Frankia strains (one isolated from an Alnus-Frankia symbiosis and two from Casuarina-Frankia associations) were investigated. All three strains responded to the addition of nickel with an increase in hydrogen uptake. Additional nickel did not affect nitrogenase activity, however evolved hydrogen was detected in Frankia KB5 in the absence of additional nickel, indicating that hydrogenase was not active. No increase in the processing rate of the hydrogenase large subunit was found with increasing nickel concentrations for any of the strains, indicating that the strategy for regulating hydrogenase in Frankia is different from that in other microorganisms. Received: 23 April 2001 / Accepted: 29 May 2001     

Frankia KB5 Possesses a Hydrogenase Immunologically Related to Membrane-Bound [NiFe]-Hydrogenases

The immunological relationship of the hydrogenase in Frankia KB5 to hydrogenases in other microorganisms was investigated using antisera raised against holo-[NiFe]-hydrogenases isolated from Alcaligenes latus, Azotobacter vinelandii, Ralstonia eutropha, and the small and large hydrogenase subunits from Bradyrhizobium japonicum. The antisera raised against the A. latus, R. eutropha, and B. japonicum (large subunit) polypeptides were found to recognize two polypeptides, corresponding to the unprocessed and processed forms of the hydrogenase subunit in Frankia KB5. None of the antisera, including the antibodies produced against the small hydrogenase subunit isolated from B. japonicum, recognized any polypeptide related to the small hydrogenase subunit in Frankia KB5. An immunogold localization study of the intracellular distribution of hydrogenase in Frankia KB5, with the cryo-section technique, showed that labeling in the membrane of both hyphae and vesicles was positively correlated with hydrogenase activity. Received: 6 November 2000 / Accepted: 18 December 2000     

The membrane-bound hydrogenase of Alcaligenes eutrophus: II. Localization and immunological comparison with other hydrogenase systems

Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing. Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane.The membrane-bound hydrogenases of A. eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas, Alcaligenes and Aquaspirillum. Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotrophicus GZ 29 was found only in a few hydrogen-oxidizing bacteria. Material cross-reacting to the soluble hydrogenase of A. eutrophus was detected in strains of A. eutrophus and A. ruhlandii only.Comparison of the membrane-bound hydrogenase of A. eutrophus, P. pseudoflava and X. autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only. The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.     

Structural and gene expression analyses of uptake hydrogenases and other proteins involved in nitrogenase protection in Frankia

The actinorhizal bacterium Frankia expresses nitrogenase and can therefore convert molecular nitrogen into ammonia and the by-product hydrogen. However, nitrogenase is inhibited by oxygen. Consequently, Frankia and its actinorhizal hosts have developed various mechanisms for excluding oxygen from their nitrogen-containing compartments. These include the expression of oxygen-scavenging uptake hydrogenases, the formation of hopanoid-rich vesicles, enclosed by multi-layered hopanoid structures, the lignification of hyphal cell walls, and the production of haemoglobins in the symbiotic nodule. In this work, we analysed the expression and structure of the so-called uptake hydrogenase (Hup), which catalyses the in vivo dissociation of hydrogen to recycle the energy locked up in this ‘waste’ product. Two uptake hydrogenase syntons have been identified in Frankia: synton 1 is expressed under free-living conditions while synton 2 is expressed during symbiosis. We used qPCR to determine synton 1 hup gene expression in two Frankia strains under aerobic and anaerobic conditions. We also predicted the 3D structures of the Hup protein subunits based on multiple sequence alignments and remote homology modelling. Finally, we performed BLAST searches of genome and protein databases to identify genes that may contribute to the protection of nitrogenase against oxygen in the two Frankia strains. Our results show that in Frankia strain ACN14a, the expression patterns of the large (HupL1) and small (HupS1) uptake hydrogenase subunits depend on the abundance of oxygen in the external environment. Structural models of the membrane-bound hydrogenase subunits of ACN14a showed that both subunits resemble the structures of known [NiFe] hydrogenases (Volbeda et al. 1995), but contain fewer cysteine residues than the uptake hydrogenase of the Frankia DC12 and Eu1c strains. Moreover, we show that all of the investigated Frankia strains have two squalene hopane cyclase genes (shc1 and shc2). The only exceptions were CcI3 and the symbiont of Datisca glomerata, which possess shc1 but not shc2. Four truncated haemoglobin genes were identified in Frankia ACN14a and Eu1f, three in CcI3, two in EANpec1 and one in the Datisca glomerata symbiont (Dg).     

<Emphasis Type="Italic">Escherichia coli</Emphasis> hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities

In the past, it has been difficult to discriminate between hydrogen synthesis and uptake for the three active hydrogenases in Escherichia coli (hydrogenase 1, 2, and 3); however, by combining isogenic deletion mutations from the Keio collection, we were able to see the role of hydrogenase 3. In a cell that lacks hydrogen uptake via hydrogenase 1 (hyaB) and via hydrogenase 2 (hybC), inactivation of hydrogenase 3 (hycE) decreased hydrogen uptake. Similarly, inactivation of the formate hydrogen lyase complex, which produces hydrogen from formate (fhlA) in the hyaB hybC background, also decreased hydrogen uptake; hence, hydrogenase 3 has significant hydrogen uptake activity. Moreover, hydrogen uptake could be restored in the hyaB hybC hycE and hyaB hybC fhlA mutants by expressing hycE and fhlA, respectively, from a plasmid. The hydrogen uptake results were corroborated using two independent methods (both filter plate assays and a gas-chromatography-based hydrogen uptake assay). A 30-fold increase in the forward reaction, hydrogen formation by hydrogenase 3, was also detected for the strain containing active hydrogenase 3 activity but no hydrogenase 1 or 2 activity relative to the strain lacking all three hydrogenases. These results indicate clearly that hydrogenase 3 is a reversible hydrogenase.     

A cytoplasmic nickel-iron hydrogenase with high specific activity from Desulfovibrio multispirans sp. N., a new species of sulfate reducing bacterium

A hydrogenase from a new species of sulfate reducing bacterium has been isolated and characterized. In contrast to other hydrogenases isolated from Desulfovibrio, this enzyme is found in the cytoplasmic fraction rather than in the periplasm. The specific activity of the enzyme, as measured in the hydrogen evolution assay, is twice as high as the specific activity of the hydrogenase from D. gigas. It also differentiates itself from the periplasmic Desulfovibrio hydrogenases by being more active in the hydrogen evolution rather than in the hydrogen uptake assay. The enzyme was shown to contain 0.9 atoms of nickel, 11 atoms of iron and 10 atoms of labile sulfide per mole of enzyme. It exhibits an unusually low intensity of the g = 2.31 nickel EPR signal in the isolated enzyme but shows a normal intensity for the g = 2.19 nickel EPR signal when reduced under hydrogen.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme.

In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing.     

Diversity of Cyanobacterial Hydrogenases, a Molecular Approach

In an effort to elucidate the diversity of cyanobacterial hydrogenases, we used a molecular approach. Filamentous strains from a broad range of sources were screened for the presence of hup (uptake hydrogenase), xisC (rearrangement within hupL), and hox (bidirectional hydrogenase) genes. As expected, an uptake hydrogenase seems to be present in all N₂-fixing cyanobacteria. On the other hand, no evidence was found for the presence of a conventional bidirectional enzyme in several strains. Similarly, the presence of xisC is not a characteristic shared by all the heterocyst-forming cyanobacteria. Although tempting, it is not possible to establish a correlation between the presence/absence of the bidirectional hydrogenase and the occurrence of xisC. The natural molecular variation of hydrogenases in cyanobacteria is certainly a field to explore, both to understand the physiological functions of the respective enzymes and to identify a genetic background to be used when constructing a strain for photobiological H₂ production in a bioreactor. Received: 3 November 1999 / Accepted: 8 December 1999     

Genetic diversity and amplification of different clostridial [FeFe] hydrogenases by group-specific degenerate primers

Aims: The aim of this study was to explore and characterize the genetic diversity of [FeFe] hydrogenases in a representative set of strains from Clostridium sp. and to reveal the existence of neither yet detected nor characterized [FeFe] hydrogenases in hydrogen‐producing strains. Methods and Results: The genomes of 57 Clostridium strains (34 different genotypic species), representing six phylogenetic clusters based on their 16S rRNA sequence analysis (cluster I, III, XIa, XIb, XIV and XVIII), were screened for different [FeFe] hydrogenases. Based on the obtained alignments, ten pairs of [FeFe] hydrogenase cluster‐specific degenerate primers were newly designed. Ten Clostridium strains were screened by PCRs to assess the specificity of the primers designed and to examine the genetic diversity of [FeFe] hydrogenases. Using this approach, a diversity of hydrogenase genes was discovered in several species previously shown to produce hydrogen in bioreactors: Clostridium sartagoforme, Clostridium felsineum, Clostridium roseum and Clostridium pasteurianum. Conclusions: The newly designed [FeFe] hydrogenase cluster‐specific primers, targeting the cluster‐conserved regions, allow for a direct amplification of a specific hydrogenase gene from the species of interest. Significance and Impact of the Study: Using this strategy for a screening of different Clostridium ssp. will provide new insights into the diversity of hydrogenase genes and should be a first step to study a complex hydrogen metabolism of this genus.     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.     

Distribution analysis of hydrogenases in surface waters of marine and freshwater environments

Background

Surface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H₂ evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H₂ metabolism in marine environments is nitrogen fixation.

Principal Findings

We analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H₂ uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean.

Significance

This study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H₂-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles.     

Genomic Analysis Reveals Multiple [FeFe] Hydrogenases and Hydrogen Sensors Encoded by Treponemes from the H<Subscript>2</Subscript>-Rich Termite Gut

We have completed a bioinformatic analysis of the hydrogenases encoded in the genomes of three termite gut treponeme isolates: hydrogenotrophic, homoacetogenic Treponema primitia strains ZAS-1 and ZAS-2, and the hydrogen-producing, sugar-fermenting Treponema azotonutricium ZAS-9. H₂ is an important free intermediate in the breakdown of wood by termite gut microbial communities, reaching concentrations in some species exceeding those measured for any other biological system. The spirochetes encoded 4, 8, and 5 [FeFe] hydrogenase-like proteins, identified by their H domains, respectively, but no other recognizable hydrogenases. The [FeFe] hydrogenases represented many sequence families previously proposed in an analysis of termite gut metagenomic data. Each strain encoded both putative [FeFe] hydrogenase enzymes and evolutionarily related hydrogen sensor/transducer proteins likely involved in phosphorelay or methylation pathways, and possibly even chemotaxis. A new family of [FeFe] hydrogenases (FDH-Linked) is proposed that may form a multimeric complex with formate dehydrogenase to provide reducing equivalents for reductive acetogenesis in T. primitia. The many and diverse [FeFe] hydrogenase-like proteins encoded within the sequenced genomes of the termite gut treponemes has enabled the discovery of a putative new class of [FeFe] hydrogenase proteins potentially involved in acetogenesis and furthered present understanding of many families, including sensory, of H domain proteins beyond what was possible through the use of fragmentary termite gut metagenome sequence data alone, from which they were initially defined.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H₂ consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ consumption in the dark. The ability of the Hox⁺ mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO₂, H₂, light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Dual organism design cycle reveals small subunit substitutions that improve [NiFe] hydrogenase hydrogen evolution

Background

Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii “Deep ecotype” that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity.

Results

We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions.

Conclusions

Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.

     

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

Background  

The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either hupW or hoxW, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Nostoc sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.