ENZYME-SUBSTRATE BINDING INTERACTIONS OF NADPH-CYTOCHROME-P-450 OXIDOREDUCTASE CHARACTERIZED WITH PH AND ALTERNATE SUBSTRATE INHIBITOR STUDIES

The pH dependence of the kinetic parameters for the reaction catalyzed by NADPH-cytochrome P-450 oxidoreductase (P-450R) has been determined, using various substrates and inhibitors. All V(max) and (V/K) profiles show pK(a)s of 6.2-7.3, for an acidic group that is preferentially unprotonated for catalysis, and of 8.1-9.6, for a basic group that is preferentially protonated for catalysis. The presence of the wrong ionization state for both of these groups is tolerated more at lower ionic strength (300 mM) than at higher ionic strength (850 mM). Ionization of the basic group has a more pronounced effect on binding of substrate (cytochrome c or dichloroindophenol) than on catalysis, since ionization has only a 2-fold effect on V(max) with cytochrome c, and only a 5-fold effect on V(max) with dichloroindophenol, while (V/K) for both substrates continues to drop at high pH with no sign of reaching a plateau. Therefore, this basic group affects predominantly substrate binding and, to a lesser extent, catalysis. It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol binding site, near the FMN prosthetic group. The NADP+ pK(i) profile shows a pK(a) of 5.95 for the 2'-phosphate of NADP+, which is bound to P-450R as the dianion, and a pK(a) of 9.53 for an enzyme group that must be protonated in order to bind NADP+. Removal of the 2'-phosphate of NADPH leads to a loss of 5.0 kcal/mol of ground-state and 6.0 kcal/mol of transition-state binding energy, while removal of the 2'-phosphate of NADP+ leads to a loss of 4.7 kcal/mol of ground-state binding energy. Thus, the 2'-phosphate is providing roughly 5 kcal/mol of uniform binding energy, resulting from all of the enzyme interactions with this group. The (V/K)cytc pH profile at 300 mM ionic strength fits best to a model assuming less-than-or-equal-to 2 lysines with pK(a)s of 10.6 that are involved in binding interactions between P-450R and cytochrome c. There is also a group with a pK(a) of 7.27 that must be unprotonated for cytochrome c to bind to P-450R. These binding interactions between cytochrome c and P-450R are not significant at higher ionic strength (850 mM).