Intermediates in the reaction of substrate-free cytochrome P450(cam) with peroxy acetic acid

Freeze-quenched intermediates of substrate-free cytochrome Fe-57-P450(cam) in reaction with peroxy acetic acid as oxidizing agent have been characteri zed by EPR and Mossbauer spectroscopy. After 8 ms of reaction time the reac tion mixture consists of similar to 90%, of ferric low-spin iron with g-fac tors and hyperfine parameters of the starting material; the remaining simil ar to 10% are identified as a free radical (S' = 1/2) by its EPR and as an iron(IV) (S=1) species by its Mossbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mossbauer and EPR-spectra e xhibit 100% of the starting material. We note that the spin-Hamiltonian ana lysis of the spectra of the 8 ms reactant clearly reveals that the two para magnetic species, e,g, the ferryl (iron(IV)) species and the radical, are n ot exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr-96) into a tyrosine radical (Tyr(.)-96), and the iron(III) center of substrate-free P450(cam) to iron(IV), (C) 2000 Federation of European Biochemical Societi es. Published by Elsevier Science B.V. All rights reserved.