title: Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues.
abstract: Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.
authors.affiliation: Department of Biochemistry, Kagawa Medical School, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tsubaki
authors.forename: A
authors.initials: A
authors.lastname: Hiwatashi
authors.forename: Y
authors.initials: Y
authors.lastname: Ichikawa
authors.forename: H
authors.initials: H
authors.lastname: Hori
mesh_terms: D000302:Adrenal Cortex / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006420:Hemeproteins / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013367:Submitochondrial Particles / Q000378:metabolism
chemical_list: D006420:Hemeproteins; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol