NMR studies of cytochrome P-450scc. Effects of steroid binding on water proton access to the active site of the ferric enzyme.
Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450 (-450scc) 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 Fe3+ center, and the long electron spin relaxation time of the heme iron, tau s approximately 0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is approximately 2.5 A, 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 approximately 4 A, 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 approximately 2.7 A is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450scc with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, or (20R)-20-phenyl-5-pregnene-3 beta,20-diol, also yielded distances of approximately 2.5 A 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Jacobs
R E
RE
Department of Physiology and Biophysics, University of California, Irvine 92717.
Singh
J
J
Vickery
L E
LE
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008928:Mitochondria / Q000201:enzymology; D011485:Protein Binding; D013816:Thermodynamics; D014867:Water
D014867:Water; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme