title: Analyses of the large subunit histidine-rich motif expose an alternative proton transfer pathway in [NiFe] hydrogenases.
abstract: 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.
authors.affiliation: Department of Biotechnology, University of Szeged, Szeged, Hungary.
authors.forename: Emma
authors.initials: E
authors.lastname: Szőri-Dorogházi
authors.forename: Gergely
authors.initials: G
authors.lastname: Maróti
authors.forename: Milán
authors.initials: M
authors.lastname: Szőri
authors.forename: Andrea
authors.initials: A
authors.lastname: Nyilasi
authors.forename: Gábor
authors.initials: G
authors.lastname: Rákhely
authors.forename: Kornél L
authors.initials: KL
authors.lastname: Kovács
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019295:Computational Biology; D003198:Computer Simulation; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006864:Hydrogenase / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011522:Protons; D016415:Sequence Alignment; D020616:Thiocapsa roseopersicina / Q000201:enzymology*
chemical_list: D021122:Protein Subunits; D011522:Protons; D006639:Histidine; C043296:nickel-iron hydrogenase; D006864:Hydrogenase