Electronic and stereochemical characterizations of intermediates in the photolysis of ferric cytochrome P450scc nitrosyl complexes. Effects of cholesterol and its analogues on ligand binding structures.
Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric cytochrome P450scc was examined by EPR spectroscopy to elucidate the stereochemical interaction between heme-bound ligand and side-chain of cholesterol or its hydroxylated analogues at the substrate-binding site. The photoproducts of the NO complexes trapped at 5 K exhibited new EPR absorptions providing information on the steric crowding of the distal heme moiety. Without substrate, the photoproduct exhibited a broad EPR absorption at g-8 due to magnetic dipole-dipole interaction between the photo-dissociated NO (S = 1/2) and the ferric iron (S = 5/2). This indicates that the photo-dissociated NO can move far away from the heme iron in the less restricted distal heme moiety of the substrate-free cytochrome P450scc. In the presence of substrates, such as cholesterol, 20(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 25-hydroxycholesterol, the EPR spectra of the photoproducts exhibited many variations having broad g-8 absorptions and/or the widespread signals together with zero-field absorption. Among the steroid complexes used, 20(S)-hydroxycholesterol complex exhibited a conspicuously widespread EPR signal with a distinct zero-field absorption due to a spin-coupled interaction between the ferric iron (S = 5/2) and the photolyzed NO (S = 1/2). These results indicate that the 20(S)-hydroxycholesterol complex has restricted substrate-binding structure and that the hydroxylation of the cholesterol side-chain at the 22R position is necessary to proceed the side-chain cleavage reaction properly in cytochrome P450scc.
Hori
H
H
Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Japan.
Masuya
F
F
Tsubaki
M
M
Yoshikawa
S
S
Ichikawa
Y
Y
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006888:Hydroxycholesterols / Q000378:metabolism; D006900:Hydroxylation; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism*; D010782:Photolysis; D013237:Stereoisomerism; D013696:Temperature
D006888:Hydroxycholesterols; D008024:Ligands; D009569:Nitric Oxide; D002784:Cholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme