NMR Studies of Cytochrome P-450_(scc). Effects of Steroid Binding on Water Proton Access to the Active Site of the Ferric Enzyme

Abstract

Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450(P-450_(scc)) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe^(3+) center, and the long electron spin relaxation time of the heme iron, T_s~0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is ~2.5 Å, a distance consistent with a water molecule binding directly to the heme iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to ~4 Å, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the Complex with (22R)-22-hydroxycholesterol, a distance of ~2.7 Å is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450_(scc) with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3β-ol, or (20R)-20-phenyl-5- pregnene-3β,20-diol, also yielded distances of ~2.5 Å and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand. In contrast to cytochrome P-450_(cam), in which water is excluded from the heme center in the substrate complex [Griffin, B. W., & Peterson, J. A. (1975) J. Biol. Chem. 250, 6445-6451; Philson, S. B., Debrunner, P. G., Schmidt, P. G., & Gunsalus, I. C. (1979) J. Biol. Chem. 254, 10173-10179], protons have rapid access to regions near the active site of several steroid complexes of P-450_(scc). This suggests that the active site of P-450_(scc) may be open to solvent and that solvent water molecules, rather than acid/base groups in the active site, may provide the protons required during the monooxygenation reaction cycle.

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