Cytochrome P450_cam and its complexes, Mössbauer parameters of the heme iron

Mössbauer 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 Mössbauer 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 Mössbauer 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, Fe³⁺-O^-₂, model proposed for oxyhemoglobin (Weiss, J. J. (1964) Nature 202, 83-84). Reduced P450_cam and its complexes, oxyP450_cam and P450_cam·CO, are all found to be analogous in some respects to the corresponding hemoglobin complexes.