Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450 cam ‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide

The hydroperoxo iron(III) intermediate P450 cam Fe III –OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450 cam , could be transiently observed in the peroxo‐shunt oxidation of the substrate‐free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpd 0 enabled us to kinetically examine the formation and reactivity of P450 cam Fe III –OOH species as a function of varying reaction conditions, such as pH, and concentration of H 2 O 2 , camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate‐free form of P450 cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid–base catalyst and is mainly governed by the ability of H 2 O 2 to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p ${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$ . Notably, no spectroscopic evidence for the formation of either Cpd I or Cpd II as products of heterolytic or homolytic OO bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1 R )‐camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe III –OOH intermediate in the oxidation of the natural substrate of P450 cam .