Highly purified microsomal P-450: The oxyferro intermediate stabilized at low temperature

Abstract

The oxyferro intermediate of highly purified microsomal P-450 from rabbit liver was formed and stabilized at −30°C in a mixture of aqueous buffer and glycerol ( 1 1 ). Absolute and difference (Fe 2·+O 2-Fe 3+) spectra of this intermediate appear to be very similar to those obtained under either steady state kinetics or stopped flow conditions on the same cytochrome as well as on bacterial P-450 cam. (Absolute and difference spectra present maxima at 420 and 557–558 nm and a broad maximum at 442 nm respectively). As temperature increases the oxyferro intermediate autoxidizes and ferric cytochrome P-450 is restored. This reaction appears to follow biphasic first order kinetics. The rate constant of both phases decreases with temperature and increases with protons concentrations.