Cytochrome P450 cam and its complexes, Mo¨ssbauer parameters of the heme iron

Abstract

Mo¨ssbauer spectroscopy has been used to study the heme iron in various states of cytochrome P450 cam from the camphor-hydroxylating system of the bacterium Pseudomonas putida. Native, camphor-free P450 cam contains low-spin ferric iron, part of which (approx. 50–70%) is converted to the high-spin ferric state upon addition of camphor. The Mo¨ssbauer spectra of the camphor-free enzyme ( S = 1/2) and of the high-spin component ( S = 5/2) of the camphor complex have been successfully simulated using a model based on crystal-field theory and simple covalency considerations. The native low-spin ferric state of P450 cam forms a complex with 2-phenylimidazole, with small changes in the g values and Mo¨ssbauer spectra. These changes can be accounted for consistently in the crystal-field model referred to above. The addition of putidaredoxin to the camphor-complexed, oxidized P450 cam decreases the intensity of the high-spin component and changes its quadrupole splitting. The reduced form of P450 cam contains high-spin ferrous iron, both in the presence and absence of camphor. The complex of reduced P450 cam with molecular oxygen is diamagnetic and has a combination of quadrupole splitting and isomer shift that is unusual for a ferrous complex, but strongly resembles that of oxyhemoglobin. These results are compatible with the bound superoxide, Fe 3+-O − 2, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83–84). Reduced P450 cam and its complexes, oxy P450 cam and P450 cam ·CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes.