CONFORMATIONAL-ANALYSIS OF MITOCHONDRIAL AND MICROSOMAL CYTOCHROME-P-450 BY RESONANCE RAMAN-SPECTROSCOPY

Mitochondrial and microsomal cytochromes P-450(SCC) and P-450(LM2) in the ferric substrate-free and substrate-bound states were studied by resonance Raman spectroscopy. In the spectra of cytochrome P-450(SCC) two conformational states (A and B) were detected, each of them constituting an equilibrium between a six-coordinated low-spin and a high-spin form. Both the conformational and the spin equilibria are pH- and temperature-dependent, which is in line with previously published results [Lange, R., Larroque, C., & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73)]. On the basis of well-resolved resonance Raman spectra, measured at different pH and temperatures, these equilibria were analyzed quantitatively. Both low-spin configurations of A and B exhibit different band patterns in the spin state marker band region, indicating differences in the active-site structures. While in the high-spin configuration of state A the heme iron remains weakly bound by a sixth ligand, the high-spin form of state B is five-coordinated, Binding of cholesterol to cytochrome P-450(SCC) causes a significant population of the high-spin forms, particularly of state A (62%). On the other hand, binding of 22R-hydroxycholesterol to the substrate-free enzyme leaves the overall spin equilibrium largely unchanged, i.e., six-coordinated low spin (76% A and 24% B). In both substrate-bound complexes, interactions between the substrate and the heme lead to small but distinct differences in the resonance Raman spectra of the low-spin form of state A. In contrast to cytochrome P-450(SCC), the resonance Raman spectra of microsomal cytochrome P-450(LM2) provide no indications for multiple conformers at 22 degrees C. Binding of benzphetamine causes a partial conversion from the six-coordinated low-spin to a high-spin state (28%) in which a sixth ligand may still weakly interact with the heme iron, similar to the case of the HS species of state A in cytochrome P-450(SCC). The comparison of the results obtained for both enzymes indicate that at ambient temperature the protein structure, at least in the heme pocket and the substrate binding site, is significantly more flexible in cytochrome P-450(SCC). This conformational flexibility may be related to the ability to bind the sterically demanding natural substrates and may control the product specificity of the catalytic process.