Kinetics of Cytochrome P-450 Reduction: Evidence for Faster Reduction of the High-Spin Ferric State

Results are presented that support our hypothesis [Backes, W. L., Sligar, S. G., & Schenkman, J. B. (1980) Biochem. Biophys. Res. Commun. 97, 860-867] that the multiphasic reduction kinetics of cytochrome P-450 are, in part, due to the spin equilibrium of the ferric hemoprotein. The disappearance of the high-spin charge-transfer band at 650 nm during reduction of the hemoprotein by NADPH was fast, exhibiting a rate constant greater than that of the fast phase of reduction measured by formation of the carbon monoxide adduct. In contrast, the disappearance of the ferric low-spin form of the cytochrome was at a considerably slower rate. A mathematical expression of the fractional content of high-spin cytochrome P-450 was obtained by comparing the ratio of the initial rate of change in the fraction of total oxidized cytochrome remaining to the initial rate of change in the fraction of high-spin ferric P-450 remaining. Results supporting the model were obtained by using both microsomes and purified cytochrome P-450 RLM5. The calculation from experimental data yielded results that were similar to those obtained by different extrapolation methods used for estimation of the amount of high-spin cytochrome P-450, supporting further the proposed relationship between the spin equilibrium and the reduction kinetics of this hemoprotein.