title: H-tunneling in the multiple H-transfers of the catalytic cycle of morphinone reductase and in the reductive half-reaction of the homologous pentaerythritol tetranitrate reductase.
abstract: The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.
authors.affiliation: Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
authors.forename: Jaswir
authors.initials: J
authors.lastname: Basran
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Harris
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sutcliffe
authors.forename: Nigel S
authors.initials: NS
authors.lastname: Scrutton
mesh_terms: D001426:Bacterial Proteins; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003512:Cyclohexanones / Q000378:metabolism; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003512:Cyclohexanones; D005415:Flavins; D009243:NAD; C028215:2-cyclohexen-1-one; D005486:Flavin Mononucleotide; D006859:Hydrogen; D003903:Deuterium; D010088:Oxidoreductases; C087820:morphinone reductase; C098939:pentaerythritol tetranitrate reductase