Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study.
Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.
Tsubaki
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Hiwatashi
A
A
Ichikawa
Y
Y
D000302:Adrenal Cortex / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000378:metabolism
D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol