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.
Highlights
Catalyzed reaction rate [1]
In morphinone reductase (MR), both the reaction rates and the kinetic isotope effect (KIE) are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike pentaerythritol tetranitrate (PETN) reductase, is gated by thermally induced vibrations in the protein
Spectral changes observed in the reductive half-reactions of MR with a 10-fold excess of NADH were previously shown to fit to a two-step kinetic model: A 3 B 3 C, consistent with these assignments, where A is the oxidized enzyme, B is an enzymecoenzyme charge-transfer intermediate, and C is the enzyme containing the reduced form of the flavin cofactor [36], fitting to more complex reversible kinetic models was not explored
Summary
Catalyzed reaction rate [1]. Our quest to understand the physical basis of this catalytic power is challenging and has involved sustained and intensive research efforts by many workers in the physical and life sciences (for recent reviews see Refs. 2–5). New theoretical frameworks incorporating quantum mechanical tunneling and protein motion are emerging to address the catalytic potency of enzymes. These invoke motion in the protein and/or substrate to drive the reaction (16 –19). We demonstrate in this report that hydride transfer from nicotinamide coenzyme to flavin in both enzymes occurs by quantum tunneling but that the nature of the tunneling reaction is different. Despite the similar active site architectures, this likely reflects differences in the dynamics of the enzyme scaffold in MR and PETN reductase. We show that hydride transfer from reduced flavin to the substrate 2-cyclohexenone in MR occurs by tunneling and that this reaction is concerted with proton transfer from an unidentified active site acid to the substrate unsaturated bond
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