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) profil es 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 ion ization state for both of these groups is tolerated more at lower ioni c strength (300 mM) than at higher ionic strength (850 mM). Ionization of the basic group has a more pronounced effect on binding of substra te (cytochrome c or dichloroindophenol) than on catalysis, since ioniz ation 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 pl ateau. Therefore, this basic group affects predominantly substrate bin ding and, to a lesser extent, catalysis. It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol bind ing 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-45 0R as the dianion, and a pK(a) of 9.53 for an enzyme group that must b e protonated in order to bind NADP+. Removal of the 2'-phosphate of NA DPH leads to a loss of 5.0 kcal/mol of ground-state and 6.0 kcal/mol o f transition-state binding energy, while removal of the 2'-phosphate o f 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 bi nding 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 t hat 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 unprotonat ed for cytochrome c to bind to P-450R. These binding interactions betw een cytochrome c and P-450R are not significant at higher ionic streng th (850 mM).