Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton Transfer Pathway in [NiFe] Hydrogenases

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis

Photofermentative Production of Hydrogen by Thiocapsa Roseopersicina from Simple Organic Substrates

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

Photofermentative production of hydrogen from organic acids by the purple sulfur bacterium Thiocapsa roseopersicina

A mutant strain of the anaerobic purple sulfur bacterium Thiocapsa roseopersicina, containing only nitrogenase as a functionally active enzyme for H2 generation was utilized to study the production of H2 from organic acids (acetate, pyruvate and succinate). Two types of potential substrates for H2 production, thiosulfate and salts of various organic acids, were compared under photoheterotrophic growth conditions. Thiosulfate proved to be the preferred electron donor for T. roseopersicina; the consumption of organic acids became pronounced only following depletion of the thiosulfate supply. The system is suitable for the generation of H2 from effluents of heterotrophic dark fermentation processes or waste streams rich in inorganic reduced sulfur compounds and/or simple organic acids. Copyright © 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved

The amino acids of proposed proton transfer pathway are highly conserved.

<p>T.r.: <i>Thiocapsa roseopersicina</i> HynL, A.v.: <i>Allochromatium vinosum</i> HydL, R.c.: <i>Rhodobacter capsulatus</i> HupL, M.c.: <i>Methylococcus capsulatus</i>, B.j.: <i>Bradyrhizobium japonicum</i> HupL, Rh.l.: <i>Rhizobium leguminosarum</i> HupL, R.e.: <i>Ralstonia eutropha</i> H16 HoxG, D.v.: <i>Desulfovibrio vulgaris</i> Miyazaki F HynB, D.g.: <i>Desulfovibrio gigas</i> HynB, D.f.: <i>Desulfovibrio fructosovorans</i> HynB. The upper and lower numbering refers to the large subunits of <i>T. roseopersicina</i> and <i>D. vulgaris</i> enzymes, respectively.</p

Bacterial strains.

<p>Indicated strains and plasmids are from Stratagene, La Jolla, CA, USA.</p

<i>In vivo</i> and <i>in vitro</i> hydrogenase activity of <i>Thiocapsa roseopersicina</i> HynSL enzyme.

<p><i>In vivo</i> and <i>in vitro</i> hydrogenase activity of <i>Thiocapsa roseopersicina</i> HynSL enzyme.</p

Vectors used in this study.

<p>Vectors used in this study.</p

Mutagenesis primers used for creating new mutants.

<p>Nucleotide triplets coding mutated amino acids are bold.</p