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 r esonance Raman spectroscopy. In the spectra of cytochrome P-450(SCC) t wo conformational states (A and B) were detected, each of them constit uting an equilibrium between a six-coordinated low-spin and a high-spi n form. Both the conformational and the spin equilibria are pH- and te mperature-dependent, which is in line with previously published result s [Lange, R., Larroque, C., & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73)]. On the basis of well-resolved resonance Raman spectra, m easured at different pH and temperatures, these equilibria were analyz ed quantitatively. Both low-spin configurations of A and B exhibit dif ferent band patterns in the spin state marker band region, indicating differences in the active-site structures. While in the high-spin conf iguration of state A the heme iron remains weakly bound by a sixth lig and, the high-spin form of state B is five-coordinated, Binding of cho lesterol to cytochrome P-450(SCC) causes a significant population of t he 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, interac tions between the substrate and the heme lead to small but distinct di fferences 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 multipl e conformers at 22 degrees C. Binding of benzphetamine causes a partia l conversion from the six-coordinated low-spin to a high-spin state (2 8%) in which a sixth ligand may still weakly interact with the heme ir on, similar to the case of the HS species of state A in cytochrome P-4 50(SCC). The comparison of the results obtained for both enzymes indic ate that at ambient temperature the protein structure, at least in the heme pocket and the substrate binding site, is significantly more fle xible in cytochrome P-450(SCC). This conformational flexibility may be related to the ability to bind the sterically demanding natural subst rates and may control the product specificity of the catalytic process .