Mechanism of NAD(P)H:quinone reductase: Ab initio studies of reduced flavin and combine ab initio and molecular mechanics analysis of the hydride transfer

Quinone reductase (QR1) is a flavin-adenine-dinucleotide (FAD)-containing homodimer that carries out a nicotinamide-adenine-dinucleotide-(phosphate) (NAD(P)H)-dependent, obligatory two-electron reduction of quinones. NAD(P)H and the FAD isoalloxazine ring are parallel-stacked, such that nicotinamide-C4 (C4N) and flavin N5 (N5F) are 4 Å apart, ideally positioned for hydride transfer. The proposed QR1 mechanism involves a charge transfer relay using two flavin-adjacent amino acids. We studied quantum chemically at the ab-initio RHF/STO-3G, RHF/6-31G(d) and density functional theory (DFT) B3LYP/6-31G(d) levels several x-ray structure configurations of the ring: oxidized, reduced and reduced after charge relay.; Atomic charges, three-dimensional electrostatic potentials, Highest Occupied (HOMO's) and Lowest Unoccupied (LUMO's) Molecular Orbitals were calculated. Several partially overlapping LUMO's form a path for the reducing electrons from N5F to N1F and O2F. Protonation of O2F neutralizes the ring electronegativity. There is a LUMO at His161, a residue proposed to act in the charge relay. A proton from Tyr155 is transferred to O2F, and another may be transferred from His161 to Tyr155, resulting in equalization of the ring negative charge. The reduced isoalloxazine ring is maintained planar by the protein, providing an additional reaction driving force.; Hydride transfer from NADH to FAD was analyzed using ab initio calculations, semiempirical optimizations, and molecular dynamics. The transition state was approximated by semiempirical optimization moving only the hydride. A three dimensional grid of ab initio DFT B3LYP/6-31G(d) single point energy calculations was performed around the paths to the transition state. A reaction coordinate surface with a saddle point was obtained and relaxed by optimization of the cofactors. Due to molecular strains, the energy of the transition state is too high (about 80 kcal/mol). The charge relay lowers this energy slightly, but trial amino acid side chains relaxation calculations indicate that the protein must relax to lower it further.; Quantum mechanical results were used to run CHARMM molecular dynamics relaxing the whole protein before the reaction and at the transition state. Molecular dynamics relaxation using the quantum mechanically-derived charges lowers the reaction barrier by 70 kcal/mol. Distances between important residues show that the relaxation facilitates the charge relay by diminishing the path for proton transfer.