title: The quest for a functional substrate access tunnel in FeFe hydrogenase.
abstract: We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative "static" H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme's kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lautier
authors.forename: Pierre
authors.initials: P
authors.lastname: Ezanno
authors.forename: Carole
authors.initials: C
authors.lastname: Baffert
authors.forename: Vincent
authors.initials: V
authors.lastname: Fourmond
authors.forename: Laurent
authors.initials: L
authors.lastname: Cournac
authors.forename: Juan C
authors.initials: JC
authors.lastname: Fontecilla-Camps
authors.forename: Philippe
authors.initials: P
authors.lastname: Soucaille
authors.forename: Patrick
authors.initials: P
authors.lastname: Bertrand
authors.forename: Isabelle
authors.initials: I
authors.lastname: Meynial-Salles
authors.forename: Christophe
authors.initials: C
authors.lastname: Léger
mesh_terms: D002248:Carbon Monoxide / Q000494:pharmacology; D006859:Hydrogen / Q000737:chemistry; D006864:Hydrogenase / Q000737:chemistry* / Q000502:physiology; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000502:physiology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000494:pharmacology
chemical_list: D007506:Iron-Sulfur Proteins; D002248:Carbon Monoxide; D006859:Hydrogen; C118830:iron hydrogenase; D006864:Hydrogenase; D010100:Oxygen