Conformational analysis of mitochondrial and microsomal cytochrome P-450 by resonance Raman spectroscopy.

Mitochondrial and microsomal cytochromes P-450SCC and P-450LM2 in the ferric substrate-free and substrate-bound states were studied by resonance Raman spectroscopy. In the spectra of cytochrome P-450SCC 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-450SCC 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-450SCC, the resonance Raman spectra of microsomal cytochrome P-450LM2 provide no indications for multiple conformers at 22 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)