KINETIC MECHANISM FOR THE MODEL REACTION OF NADPH-CYTOCHROME P450 OXIDOREDUCTASE WITH CYTOCHROME-C

The kinetic mechanism of NADPH-cytachrome P450 oxidoreductase (P450R) has been determined for the model reaction with cytochrome c(3+). Although initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formation of a ternary NADPH.P450R.cytochrome c(3+) complex, product and dead-end inhibition results suggest a nonclassical (two-site) ping-pong mechanism [Northrop, D. B. (1969) J. Biol. Chem. 244, 5808-5819]. This mechanism is a hybrid of the random sequential (ternary complex) and ping-pong mechanisms, since ternary complexes can form as well as intermediate, modified forms of the enzyme that can be present in the absence of any bound substrate. The complete rate equation is derived for this mechanism, and values for V-max, (VIK)(NADPH), (V/K)(cytc), and the corresponding Michaelis constants are presented in terms of microscopic rate constants along with the expected product inhibition patterns (Appendix). Inhibition by NADP(+) is competitive versus NADPH and uncompetitive versus cytochrome c(3+), while inhibition by cytochrome C2+ is competitive versus cytochrome c(3+) and noncompetitive versus NADPH. These inhibition patterns are consistent with the proposed two-site mechanism. This mechanism would give the same initial velocity patterns as the classical one-site ping-pong mechanism, but it allows for the formation of a ternary complex, with NADPH and cytochrome c(3+) reacting independently at two separate sites on P450R. The (D)(V/K)(NADPH) isotope effect is not affected by cytochrome c(3+) concentration, consistent with our assumption (in deriving the rate equation) that binding at the two sites is independent. At the high ionic strength used in this study (850 mM), the mechanism is two-site ping-pong, with the electron acceptor site itself reacting with cytochrome c(3+) in a tetra uni ping-pong manner.