Rate-limiting steps in oxidations catalyzed by rabbit cytochrome P450 1A2

Several issues regarding the rate-limiting nature of individual reaction steps in catalysis by rabbit liver cytochrome P450 (P450) 1A2 were addressed using anisoles and other substrates. Substrate binding is very fast (k > 10(6) M-1 s(-1)). Product release is not rate-limiting, as shown by the absence of bursts, placing rate-limiting steps at or before product formation. We had previously shown that the first 1-electron reduction step is fast (k > 700 min(-1)), even in the absence of ligand [Guengerich, F. P., and Johnson, W. W. (1997) Biochemistry 36, 14741-147500]. O-2 binding to ferrous P450 is fast (k greater than or equal to 10(6) M-1 s(-1)). The decay of the P450 Fe2+-substrate-O-2 complex was slow in the absence of NADPH-P450 reductase, with a first-order rate constant of 14 min(-1) at 25degreesC. During the decay, product was formed (from the substrate methacetin) in 61% theoretical yield, although this reaction requires electron transfer among P450 molecules and may not be related to normal turnover. Steady-state spectra suggest that one or more iron-oxygen complexes accumulate, representing entities between the Fe2+-O-2 complex and putative FeO3+ entity. Kinetic isotope effect experiments were done with several substrates, mainly anisoles. Apparent intrinsic deuterium isotope effects as high as 15 were measured. In all cases, the C-H bond-breaking step is at least partially rate-limiting. The isotope effects were not strongly attenuated in noncompetitive or competitive experiments, consistent with relatively rapid P450-substrate exchange, except with the active enzyme Fe-O complex. Kinetic simulations with the available data (i) are consistent with the view that C-H bond breaking is a major rate-Limiting step, (ii) demonstrate that increasing the rate of this step will affect k(cat), k(m), and kinetic hydrogen isotope effects but will only increase catalytic efficiency to a certain degree, (iii) indicate that increasing ground-state binding can increase catalytic efficiency but not k at, and (iv) suggest that nonproductive binding modes and abortive reduction of O-2 are factors that attenuate catalytic efficiency.