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 c3+ 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determined at 850 mM ionic strength [Sem, D.S., & Kasper, C. B. (1994) Biochemistry 33, 12012-12021], this mechanism is no longer valid at lower ionic strength.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 c3+ 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 c3+ 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 Vmax decreases (below 500 mM ionic strength). These results are consistent with favorable ionic interactions being important for binding NADPH and cytochrome c3+ and with product (NADP+) release becoming the rate-limiting step in Vmax at low ionic strength. Vmax decreases significantly at higher ionic strength (> 500 mM), while (V/K)NADPH decreases only slightly. The DV isotope effect is largest (2.4) at 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 DV.(ABSTRACT TRUNCATED AT 250 WORDS)