RESONANCE RAMAN INVESTIGATIONS OF ESCHERICHIA-COLI-EXPRESSED PSEUDOMONAS-PUTIDA CYTOCHROME-P450 AND CYTOCHROME-P420

High-resolution resonance Raman spectra of the ferric, ferrous, and carbonmonoxy (CO)-bound forms of wild-type Escherichia coli-expressed Pseudomonas putida cytochrome P450cam and its P420 form are reported. The ferric and ferrous species of P450 and P420 have been studied in both the presence and absence of excess camphor substrate. In ferric, camphor-bound, P450 (m(os)), the E. coli-expressed P450 is found to be spectroscopically indistinguishable from the native material. Although substrate binding to P450 is known to displace water molecules from the heme pocket, altering the coordination and spin state of the heme iron, the presence of camphor substrate in P420 samples is found to have essentially no effect on the Raman spectra of the heme in either the oxidized or reduced state. A detailed study of the Raman and absorption spectra of P450 and P420 reveals that the P420 heme is in equilibrium between a high-spin, five-coordinate (HS,5C) form and low-spin six-coordinate (LS,6C) form in both the ferric and ferrous oxidation states. In the ferric P420 state, H2O evidently remains as a heme ligand, while alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys(357) thiolate binding to the heme iron. Ferrous P420 also consists of an equilibrium between HS,5C and LS,6C states, with the spectroscopic evidence indicating that H2O and histidine are the most likely axial ligands. The spectral characteristics of the CO complex of P42O are found to be almost identical to those of a low pH of Mb. Moreover, we find that the 10-ns transient Raman spectrum of the photolyzed P420 CO complex possesses a band at 220 cm-1, which is strong evidence in favor of histidine ligation in the CO-bound state. The equilibrium structure of ferrous P420 does not show this band, indicating that Fe-His bond formation is favored when the iron becomes more acidic upon CO binding. Raman spectra of stationary samples of the CO complex of P450 reveal nu(Fe-CO) peaks corresponding to both substrate-bound and substrate-free species and demonstrate that substrate dissociation is coupled to CO photolysis. Analysis of the relative band intensities as a function of photolysis indicates that the CO photolysis and rebinding rates are faster than camphor rebinding and that CO binds to the heme faster when camphor is not in the distal pocket.