The quest for a functional substrate access tunnel in FeFe hydrogenase

• Published in: Faraday discussions Vol. 148; no. 148; pp. 385 - 407
• Main Authors: Lautier, Thomas, Ezanno, Pierre, Baffert, Carole, Fourmond, Vincent, Cournac, Laurent, Fontecilla-Camps, Juan C, Soucaille, Philippe, Bertrand, Patrick, Meynial-Salles, Isabelle, Léger, Christophe
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 01.01.2011
• Subjects: Carbon Monoxide - pharmacology; Chemical Sciences; Hydrogen - chemistry; Hydrogenase - chemistry; Hydrogenase - physiology; Inorganic chemistry; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - physiology; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Oxygen - pharmacology
• ISSN: 1359-6640; 1364-5498
• DOI: 10.1039/c004099c

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.