title: Importance of the Active Site "Canopy" Residues in an O2-Tolerant [NiFe]-Hydrogenase.
abstract: The active site of Hyd-1, an oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Escherichia coli, contains four highly conserved residues that form a "canopy" above the bimetallic center, closest to the site at which exogenous agents CO and O2interact, substrate H2binds, and a hydrido intermediate is stabilized. Genetic modification of the Hyd-1 canopy has allowed the first systematic and detailed kinetic and structural investigation of the influence of the immediate outer coordination shell on H2activation. The central canopy residue, arginine 509, suspends a guanidine/guanidinium side chain at close range above the open coordination site lying between the Ni and Fe atoms (N-metal distance of 4.4 Å): its replacement with lysine lowers the H2oxidation rate by nearly 2 orders of magnitude and markedly decreases the H2/D2kinetic isotope effect. Importantly, this collapse in rate constant can now be ascribed to a very unfavorable activation entropy (easily overriding the more favorable activation enthalpy of the R509K variant). The second most important canopy residue for H2oxidation is aspartate 118, which forms a salt bridge to the arginine 509 headgroup: its mutation to alanine greatly decreases the H2oxidation efficiency, observed as a 10-fold increase in the potential-dependent Michaelis constant. Mutations of aspartate 574 (also salt-bridged to R509) to asparagine and proline 508 to alanine have much smaller effects on kinetic properties. None of the mutations significantly increase sensitivity to CO, but neutralizing the expected negative charges from D118 and D574 decreases O2tolerance by stabilizing the oxidized resting NiIII-OH state ("Ni-B"). An extensive model of the catalytic importance of residues close to the active site now emerges, whereby a conserved gas channel culminates in the arginine headgroup suspended above the Ni and Fe.
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, U.K.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Brooke
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, U.K.
authors.forename: Rhiannon M
authors.initials: RM
authors.lastname: Evans
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, U.K.
authors.forename: Shams T A
authors.initials: ST
authors.lastname: Islam
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.
authors.forename: Gerri M
authors.initials: GM
authors.lastname: Roberts
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, U.K.
authors.forename: Sara A M
authors.initials: SA
authors.lastname: Wehlin
authors.affiliation: Research Complex at Harwell, Rutherford Appleton Laboratory , Harwell Campus, Didcot, U.K.|Department of Biochemistry, University of Oxford , Oxford, U.K.
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Carr
authors.affiliation: Research Complex at Harwell, Rutherford Appleton Laboratory , Harwell Campus, Didcot, U.K.|Department of Biochemistry, University of Oxford , Oxford, U.K.
authors.forename: Simon E V
authors.identifier: http://orcid.org/0000-0001-8922-0250
authors.initials: SE
authors.lastname: Phillips
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, U.K.
authors.forename: Fraser A
authors.identifier: http://orcid.org/0000-0001-8041-2491
authors.initials: FA
authors.lastname: Armstrong
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002245:Carbon Dioxide / Q000494:pharmacology; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006859:Hydrogen / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D020125:Mutation, Missense; D010084:Oxidation-Reduction / Q000187:drug effects; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D029968:Escherichia coli Proteins; D002245:Carbon Dioxide; D001224:Aspartic Acid; D006859:Hydrogen; D001120:Arginine; D011392:Proline; D010088:Oxidoreductases; C043296:nickel-iron hydrogenase; C029746:uptake hydrogenase; D006864:Hydrogenase; D008239:Lysine; D010100:Oxygen