EFFECT OF IONIC-STRENGTH ON THE KINETIC MECHANISM AND RELATIVE RATE LIMITATION OF STEPS IN THE MODEL NADPH-CYTOCHROME P450 OXIDOREDUCTASE REACTION WITH CYTOCHROME-C

Although the kinetic mechanism of the NADPH-cytochrome P450 oxidoreductase (P450R) reaction with cytochrome c(3+) has been determined at 850 mM ionic strength [Sem, D. S., & Kasper, C. B. (1994) Biochemistry 33, 12012-12021], this mechanism is no longer valid at lower ionic strength. At 850 mM ionic strength, the mechanism is two-site ping-pong, and reaction at the electron acceptor site is itself ping-pong. As the ionic strength is decreased below 850 mM, the initial velocity profiles begin to show curvature when cytochrome c(3+) is the varied substrate. These data are consistent with a mechanism that is still two-site ping-pong, but now with random sequential binding of two molecules of cytochrome c(3+) at the electron acceptor site. Decreasing ionic strength also causes a change in rate-limiting steps, with (V/K)(cytc) and (V/K)(NADPH) increasing while V-max decreases (below 500 mM ionic strength). These results are consistent with favorable ionic interactions being important for binding NADPH and cytochrome c(3+) and with product (NADP(+)) release becoming the rate-limiting step in V-max at low ionic strength. V-max decreases significantly at higher ionic strength (>500 mM), while (V/K)(NADPH) decreases only slightly. The V-D isotope effect is largest (2.4) al 500 mM ionic strength but decreases at both low and high ionic strength as steps other than hydride transfer become more rate-limiting. (D)(V/K)(NADPH) also decreases at both low and high ionic strength, but to a lesser extent than V-D. These data are most consistent with a model in which hydride transfer is a significant rate-limiting step in the ionic strength range of 200-750 mM, but since the intrinsic isotope effect is greater than or equal to 4.3, hydride transfer is not entirely rate limiting. At lower ionic strength, NADP(+) release becomes rate-limiting, and at higher ionic strength, some step other than hydride transfer becomes rate-limiting. This step occurs after NADPH binding, but before hydride transfer, so it probably represents a conformational change that must occur prior to hydride transfer. Finally, a novel approach is presented for determining lower limit estimates of the intrinsic isotope effect, the forward commitment to catalysis (C-f), and the V ratio (C-vf). This method is applicable to enxymes with ping-pong mechanisms and small reverse commitments to catalysis (C-r).