Probing the molecular determinants of coenzyme selectivity in the P450 BM3 FAD/NADPH domain

Bacillus megaterium P450 BM3 (BM3) is an NAD(P)H-binding diflavin reductase exhibiting substantial coenzyme specificity for NADPH over NADH. The side chains of serine 965, arginine 966 and lysine 972 in its FAD-binding domain bind the NADPH 2'-phosphate. Optical, kinetic and thermodynamic properties of S965A, R966A and K972A FAD domains were analyzed singly and combined with the FAD-shielding W1046A mutation. Steady-state and stopped-flow kinetic studies demonstrated substantially decreased NADPH affinity versus wild-type (WT) FAD domain (146-fold for the S965A K(d)). Considerable catalytic efficiency increases (the ratio of specificity constants, k(cat)/K(m), for the coenzymes) with NADH were observed for each point mutant over WT (570-fold in K972A), along with increased rates of NADH-dependent FAD reduction (k(lim) elevated 5.2-fold in R966A). In combination with W1046A, considerable (37 to 56-fold) improvements over WT were seen in the k(lim) parameters with NADH for all double mutants. Each 2'-phosphate binding point mutant produced large increases in FAD potential (111 mV in R966A), despite large distances between these residues and the FAD isoalloxazine ring (18-21 A), suggesting long range conformational influences on FAD environment. The W1046A/K972A mutant abolished NADPH selectivity (8340-fold coenzyme selectivity switch towards NADH), with ramifications for BM3's biotechnological exploitation using the cheaper NADH coenzyme.