title: Experimental approaches to kinetics of gas diffusion in hydrogenase.
abstract: Hydrogenases, which catalyze H(2) to H(+) conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H(2) toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.
authors.affiliation: Centre National de la Recherche Scientifique, Institut de Biologie Structurale et Microbiologie, Unité Propre de Recherche 9036, Unité de Bioénergétique et Ingénierie des Protéines, Marseille, France.
authors.forename: Fanny
authors.initials: F
authors.lastname: Leroux
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dementin
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Burlat
authors.forename: Laurent
authors.initials: L
authors.lastname: Cournac
authors.forename: Anne
authors.initials: A
authors.lastname: Volbeda
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Champ
authors.forename: Lydie
authors.initials: L
authors.lastname: Martin
authors.forename: Bruno
authors.initials: B
authors.lastname: Guigliarelli
authors.forename: Patrick
authors.initials: P
authors.lastname: Bertrand
authors.forename: Juan
authors.initials: J
authors.lastname: Fontecilla-Camps
authors.forename: Marc
authors.initials: M
authors.lastname: Rousset
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002248:Carbon Monoxide / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005740:Gases / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry*; D006864:Hydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D005740:Gases; D002248:Carbon Monoxide; D006859:Hydrogen; D006864:Hydrogenase