Tryptophan 697 Modulates Hydride and Interflavin Electron Transfer in Human Methionine Synthase Reductase

Abstract

Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698Δ, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698Δ and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2',5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.