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 temperaturedependent 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 ␣/␤ unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperaturedependent reaction rates, again consistent with groundstate 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.Enzymes are phenomenal catalysts that can achieve rate enhancements of up to ϳ21 orders of magnitude over the uncatalyzed 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). Recent years have witnessed new activity in this area and extended our theoretical understanding beyond the shortcomings of transition state theory (6) to include roles for protein "motion" (7, 8), low barrier hydrogen bonds (e.g. Refs. 9 -11), active site preorganization (e.g. reviewed in Refs. 4 and 12), and quantum mechanical tunneling (for recent reviews see Refs. 13-15). 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 subs...

Atomic Resolution Structures and Solution Behavior of Enzyme-Substrate Complexes of Enterobacter cloacae PB2 Pentaerythritol Tetranitrate Reductase

“…k Ϫ2 ϳ0; see Ref. 15 for more detailed discussion), and similar observations were made for the W102Y and W102F enzymes by global fitting of spectral data sets obtained in stopped-flow studies of the reductive half-reaction (data not shown). Consistent with the kinetic model proposed for wild-type enzyme (Scheme 1), charge-transfer complex (E-NADPH) formation in the mutant enzymes is dependent on NADPH concentration and is reversible (Fig.…”

“…The reaction of PETN reductase comprises two half-reactions: in the reductive half-reaction, enzyme is reduced by NADPH to yield the dihydroquinone form of the enzyme-bound FMN, and in the oxidative half-reaction the flavin is oxidized by the nitro-containing explosive substrates or cyclic enone substrates. A detailed kinetic mechanism based on stopped-flow data has been proposed (14), and recently hydride transfer in the reductive half-reaction was shown to proceed by quantum mechanical tunneling (15).…”

Contribution of Flavin Covalent Linkage with Histidine 99 to the Reaction Catalyzed by Choline Oxidase

“…Tunneling mechanisms have been shown in a wide array of cofactor-dependent enzymes, including flavoenzymes. Examples of flavoenzymes in which the tunneling mechanisms have been demonstrated include morphinone reductase (29,30), pentaerythritol tetranitrate reductase (29), glucose oxidase (31)(32)(33), and choline oxidase (34). Mechanistic data on Class 2 dihydroorotate dehydrogenases, also with a flavin cofactor (FMN) covalently linked to the protein moiety (35,36), could only propose a mechanism that is either stepwise or concerted with significant quantum mechanical tunneling for the hydride transfer from C6 and the deprotonation at C5 in the oxidation of dihydroorotate to orotate (37).…”

Role of Active Site Residues and Solvent in Proton Transfer and the Modulation of Flavin Reduction Potential in Bacterial Morphinone Reductase

“…1, A and B) is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexen-1-one and NADH at saturating concentrations and hydride transfer from FMNH 2 to 2-cyclohexen-1-one occurs by tunneling (26). A large solvent isotope effect accompanies the oxidative half-reaction, and double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexen-1-one, and the protonation of 2-cyclohexen-1-one by an unknown donor, are coupled (26).…”

“…1A) has been studied by stopped-flow and steady-state kinetic methods (25,26). The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer (26). The oxidative half-reaction (Fig.…”