Hydrogenase in <i>Frankia</i> KB5: Expression of and relation to nitrogenase

Uptake hydrogenase is an enzyme that is beneficial for nitrogen fixation in bacteria. Recent studies have shown that Frankia sp. has two sets of uptake hydrogenase genes, organized in synton 1 and synton 2. In the present study, phylogenetic analysis of the structural subunits of hydrogenase syntons 1 and 2 showed a distinct clustering pattern between the proteins of Frankia strains that were isolated from different host plants and non-Frankia organisms. The structural subunits of hydrogenase synton 1 of Frankia sp. CpI1, Frankia alni ACN14a, and F. alni AvCI1 were grouped together while those of Frankia spp. CcI3, KB5, UGL140104, and UGL011102 formed another group. The structural subunits of hydrogenase synton 2 of F. alni ACN14a and Frankia spp. CcI3 and BCU110501 grouped together, but those of Frankia spp. KB5 and CpI1, F. alni ArI3, and F. alniAvCI1 comprised a separate group. The structural subunits of hydrogenase syntons 1 and 2 of Frankia sp. EAN1pec were more closely related to those of non-Frankia bacteria, i.e., Streptomyces avermitilis and Anaeromyxobacter sp., respectively, than to those of other Frankia strains, suggesting the occurrence of lateral gene transfer between these organisms. In addition, the accessory Hyp proteins of hydrogenase syntons 1 and 2 of F. alni ACN14a and Frankia sp. CcI3 were shown to be phylogenetically more related to each other than to those of Frankia EAN1pec.

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The enzymes directly involved in hydrogen metabolism in cyanobacteria are hydrogenases and nitrogenase. Functionally, two types of hydrogenase are known: uptake hydrogenase (Hup) and bidirectional hydrogenase (Hox). Distribution of hydrogenases among 15 filamentous cyanobacteria was studied by heterologous Southern hybridizations with hupL and hoxH probes from Anabaena PCC7120 and by direct assay of in vitro hydrogenase activities. All tested 15 strains showed hybridization with the hupL probe. With hoxH probe, hybridization bands are detected in 12 of them, but not in 3 of them. Moreover, the latter have no detectable in vitro Hox activity. The hupL and/or hoxH gene from Anabaena PCC7120 were inactivated. The extracts from the hoxH- strains were completely unable to perform Na2S2O4- and methyl viologen-dependent H2 evolution. H2 uptake with PMS by extracts from N2 fixing cells was undetectable in hupL-. The hupL- and hupL-hoxH- strains produced 4-7 times higher amounts of H2 than the wild-type strains. The nitrogenase activities of these hupL- strains were not reduced compared to the wild-type. Unexpectedly, the hoxH- strains were lower in H2 production activities by 20-50% compared to the wild-type.

H2 is an ideal, clean and potentially sustainable energy carrier for the future due to its large energy content per weight, abundance and non-polluting nature. The selection of optimal H2 production technology depends on the H2-producing enzymes available. Thiocapsa roseopersicina contains a nitrogenase and several [NiFe] hydrogenases, which participate in H2 metabolism. In the present study, H2 production by the Hox1 soluble hydrogenase and the nitrogenase were investigated. The amount of H2 evolved by the nitrogenase enzyme was much higher than the amount produced by the Hox1 hydrogenase enzyme. By comparing the H2 production by nitrogenase from five short-chain organic acids (acetate, citrate, pyruvate, succinate, formate) the highest productivity of H2 (~3 times) was observed in the presence of 4 g/l pyruvate. In this case, the pyruvate consumption was 100%, the biomass growth was equal to that of the control, therefore the produced H2 derived from pyruvate.

In vivo the hydrogenase of Rhodopseudomonas capsulata is capable of recycling molecular hydrogen, which is coupled to the nitrogenase for acetylene redaction. A ferredoxin from R. capsulata can be reduced by native hydrogenase with molecular hydrogen as electron donor. The reducing power generated by H 2 -hydrogenase could couple to nitrogenase-dependent acetylene reduction via ferredoxin. A natural fraction from crude extracts of R. capsulata has been separated, which functions as an active electron carrier between H 2 -hydrogenase system and acetylene reduction reaction by native nitrogenase. A low midpoint redox potential component was identified in this natural fraction. The evidence indicates that the component effective for the coupling of electron might be a cytochrome C 3 . A methyl viologen-linked diaphorase activity specific to NADPH has been identified. The possible role of ferredoxincytochrome C 3 complex as an electron carrier system in the hydrogen evolution and hydrogen recycling process by R. capsulata was discussed,

A brief discussion is presented of recent information concerning (a) factors influencing extent of N2, loss during N2 fixation by legumes; (b) electron carriers involved in the oxyhydrogen reaction of Rhizobium japonicum bacteroids; and (c) progress made in evaluating H2 recycling advantages. A major factor determining whether H2 is evolved from legume nodules is the presence of an active uptake hydrogenase which participates in the oxidation of H2 that is evolved as a by-product of the nitrogenase reaction. The extent of H2 evolution from the nitrogenase reaction is affected by those factors that influence the nitrogenase turnover rate. These include the supply of ATP and reductant and the ratio of the Fe protein to the MoFe protein component of nitrogenase. Oxidation of H2 in Rhizobium bacteroids is catalyzed by a series of enzymes located in bacteroid membranes. In addition to the hydrogenase per se, carriers so far shown to be involved in the process include cytochromes of the b and c types a...

Reactive metalloradical intermediates have been implicated in both biological and synthetic catalyst systems for small molecule activation processes, including proton reduction and ammonia oxidation. Towards a greater mechanistic understanding of such transformations on well-defined model complexes, this thesis explores relevant H–H and N–N bond-forming reactions mediated by trivalent Fe and Ni species, as well as catalytic N–N bond cleavage mediated by an open-shell VFe bimetallic complex. First, a pair of thiolate-supported, S = ½ iron and nickel hydrides are synthesized and spectroscopically characterized at low temperatures (Chapters 2, 3). Paramagnetic iron and nickel hydrides have been proposed as catalytic intermediates of [NiFe] hydrogenase and nitrogenase, but characterization of such molecular species are limited. For both the FeIII and NiIII hydride complexes described herein, spin delocalization onto the thiolate ligand is proposed to stabilize the formal 3+ metal oxidation state. Furthermore, both the FeIII–H and NiIII–H species are demonstrated to undergo the bimolecular reductive elimination of dihydrogen upon warming, albeit with distinct activation parameters consistent with different proposed pathways for H–H bond formation. Chapter 4 expands upon the H–H bond forming chemistry demonstrated on the Ni system to demonstrate related N–N bond formation from an analogous NiIII–NH2 species, resulting in the formation of a NiII2(N2H4) complex. Given the diverse mechanistic possibilities for the overall 6e-/6H+ transformation to oxidize ammonia to dinitrogen, identification of the active M(NHx) intermediate and pathway for N–N bond formation is a central mechanistic question. While the homocoupling of M–NH2 species to form hydrazine has been hypothesized as the key N–N bond forming step in ammonia oxidation systems, stoichiometric examples of this transformation from M–NH2 complexes are rare. Lastly, Chapter 5 details the synthesis of a heterobimetallic VFe complex featuring a bridging thiolate, inspired by the structure of the VFe nitrogenase cofactor. This VFe species is demonstrated to be an active catalyst for the disproportionation of hydrazine to dinitrogen and ammonia. Notably, the heterobimetallic complex is appreciably more active than monometallic analogues of the individual V and Fe sites, suggesting that bimetallic cooperativity may facilitate the observed catalysis.