Mössbauer and EPR Study of Reaction Intermediates of Cytochrome P450

Abstract

We present a complementary Mössbauer and EPR study on reaction intermediates of substrate-free and substrate-bound cytochrome P450_cam from Pseudomonas putida prepared by the freeze-quench method from ⁵⁷Fe-labeled P450_cam using peroxy acetic acid as oxidizing agent. When reacting the substrate-free P450_cam for 8 ms reaction time the reaction mixture consists of ∼85% of ferric low-spin iron (Fe(III)) with g-factors and hyperfine parameters of the starting material; the remaining ∼15% are identified as ferryl iron (Fe(IV); S _Fe=1) by its Mössbauer signature. Parallel to the ferryl iron a tyrosine radical (S _rad=1/2) is formed. The two paramagnetic species are not exchange-coupled; however, they are close enough to significantly influence the (EPR) relaxation behavior of the radical spin. In the case of substrate-bound P450_cam only trace amounts of the tyrosine radical are formed within 8 ms (<3%); within the accuracy of Mössbauer spectroscopy (5%) iron(IV) can not be detected. The results point to Tyr-96, which is hydrogen-bonded to the substrate camphor, as the candidate for the observed tyrosine radical.