Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH.

Abstract

Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)