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+). Alth ough initial velocity studies show parallel patterns, consistent with a classical (one-site) ping-pong mechanism that precludes the formatio n of a ternary NADPH.P450R.cytochrome c(3+) complex, product and dead- end inhibition results suggest a nonclassical (two-site) ping-pong mec hanism [Northrop, D. B. (1969) J. Biol. Chem. 244, 5808-5819]. This me chanism is a hybrid of the random sequential (ternary complex) and pin g-pong mechanisms, since ternary complexes can form as well as interme diate, modified forms of the enzyme that can be present in the absence of any bound substrate. The complete rate equation is derived for thi s mechanism, and values for V-max, (VIK)(NADPH), (V/K)(cytc), and the corresponding Michaelis constants are presented in terms of microscopi c rate constants along with the expected product inhibition patterns ( Appendix). Inhibition by NADP(+) is competitive versus NADPH and uncom petitive versus cytochrome c(3+), while inhibition by cytochrome C2+ i s competitive versus cytochrome c(3+) and noncompetitive versus NADPH. These inhibition patterns are consistent with the proposed two-site m echanism. 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+) reacti ng independently at two separate sites on P450R. The (D)(V/K)(NADPH) i sotope effect is not affected by cytochrome c(3+) concentration, consi stent with our assumption (in deriving the rate equation) that binding at the two sites is independent. At the high ionic strength used in t his study (850 mM), the mechanism is two-site ping-pong, with the elec tron acceptor site itself reacting with cytochrome c(3+) in a tetra un i ping-pong manner.