title: Site saturation mutagenesis demonstrates a central role for cysteine 298 as proton donor to the catalytic site in CaHydA [FeFe]-hydrogenase.
abstract: [FeFe]-hydrogenases reversibly catalyse molecular hydrogen evolution by reduction of two protons. Proton supply to the catalytic site (H-cluster) is essential for enzymatic activity. Cysteine 298 is a highly conserved residue in all [FeFe]-hydrogenases; moreover C298 is structurally very close to the H-cluster and it is important for hydrogenase activity. Here, the function of C298 in catalysis was investigated in detail by means of site saturation mutagenesis, simultaneously studying the effect of C298 replacement with all other 19 amino acids and selecting for mutants with high retained activity. We demonstrated that efficient enzymatic turnover was maintained only when C298 was replaced by aspartic acid, despite the structural diversity between the two residues. Purified CaHydA C298D does not show any significant structural difference in terms of secondary structure and iron incorporation, demonstrating that the mutation does not affect the overall protein fold. C298D retains the hydrogen evolution activity with a decrease of k(cat) only by 2-fold at pH 8.0 and it caused a shift of the optimum pH from 8.0 to 7.0. Moreover, the oxygen inactivation rate was not affected demonstrating that the mutation does not influence O(2) diffusion to the active site or its reactivity with the H-cluster. Our results clearly demonstrate that, in order to maintain the catalytic efficiency and the high turnover number typical of [FeFe] hydrogenases, the highly conserved C298 can be replaced only by another ionisable residue with similar steric hindrance, giving evidence of its involvement in the catalytic function of [FeFe]-hydrogenases in agreement with an essential role in proton transfer to the active site.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Morra
authors.forename: Alberto
authors.initials: A
authors.lastname: Giraudo
authors.forename: Giovanna
authors.initials: G
authors.lastname: Di Nardo
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain / Q000235:genetics; D046969:Clostridium acetobutylicum / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D003545:Cysteine / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006859:Hydrogen / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D003062:Codon; D007506:Iron-Sulfur Proteins; D011522:Protons; D001224:Aspartic Acid; D006859:Hydrogen; D004247:DNA; C118830:iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine; D010100:Oxygen