The formation and decay of the oxyferrous complex of beef adrenocortical cytochrome P-450scc. Rapid-scan and stopped-flow studies.

The formation and spontaneous decay of the oxyferrous complex of purified beef adrenocortical cholesterol-bound (high spin) cytochrome P-450scc have been studied by means of rapid-scan spectrometry in the Soret region at 4 degrees C. The oxyferrous complex, the formation of which occurs within 40 ms with a Soret absorption peak at 422 nm, is unstable and decays spontaneously to the ferric cholesterol-bound cytochrome P-450scc. The rapid-scan spectra for both processes were recorded. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous complex at 418 nm, between oxyferrous complex and ferric cytochrome P-450scc at 411 nm. The kinetics of oxygen binding and spontaneous decay of the oxyferrous complex have also been studied at 4 degrees C by means of stopped-flow experiments in the pH range 5.1-8.8. The rate constant for oxygen binding is constant at 5.8 X 10(5) +/- 0.8 X 10(5) M-1 X S-1 over the pH range of the study. On the other hand, the decay process exhibited pH-dependent monophasic first-order kinetics. The rate constant for the decay appears to be influenced by an acid group with a pKa of 7.1 on the oxyferrous complex of cholesterol-bound cytochrome P-450.