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

• Published in: The Journal of biological chemistry Vol. 278; no. 45; pp. 43973 - 43982
• Main Authors: Basran, Jaswir, Harris, Richard J., Sutcliffe, Michael J., Scrutton, Nigel S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.11.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Bacterial Proteins; Catalysis; Chemical Phenomena; Chemistry, Physical; Cyclohexanones - metabolism; Deuterium; Escherichia coli - enzymology; Flavin Mononucleotide - metabolism; Flavins - metabolism; Hydrogen - metabolism; Hydrogen-Ion Concentration; Kinetics; NAD - metabolism; Oxidation-Reduction; Oxidoreductases - metabolism; Spectrophotometry; Temperature; Thermodynamics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305983200

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 α/β 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.