Solvent as a Probe of Active Site Motion and Chemistry during the Hydrogen Tunnelling Reaction in Morphinone Reductase

The reductive half‐reaction of morphinone reductase involves a hydride transfer from enzyme‐bound β‐nicotinamide adenine dinucleotide (NADH) to a flavin mononucleotide (FMN). We have previously demonstrated that this step proceeds via a quantum mechanical tunnelling mechanism. Herein, we probe the effect of the solvent on the active site chemistry. The pK_a of the reduced FMN N1 is 7.4±0.7, based on the pH‐dependence of the FMN midpoint potential. We rule out that protonation of the reduced FMN N1 is coupled to the preceding H‐transfer as both the rate and temperature‐dependence of the reaction are insensitive to changes in solution pH above and below this pK_a. Further, the solvent kinetic isotope effect is ～1.0 and both the 1° and 2° KIEs are insensitive to solution pH. The effect of the solvent’s dielectric constant is investigated and the rate of H‐transfer is found to be unaffected by changes in the dielectric constant between ～60 and 80. We suggest that, while there is crystallographic evidence for some water in the active site, the putative promoting motion involved in the H‐tunnelling reaction is insensitive to such changes.