Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of 20(R),22(R)-dihydroxycholesterol and reduced adrenodoxin.
Electron paramagnetic resonance (EPR) spectra of ferrous-nitric oxide (14NO and 15NO) cytochrome P-450scc complexed with 20(R),22(R)-dihydroxycholesterol were measured at 77 K with X-band (9.35 GHz) microwave frequency. The EPR spectra clearly showed the spin system to have rhombic symmetry (gx = 2.068, gz = 2.001, gy = 1.961, and Az = 1.89 mT for 14NO) and were distinct from those of 20(S)-hydroxycholesterol complexes. The unique nature of the 20(S)-hydroxycholesterol complexes indicates that 20(S)-hydroxycholesterol is not a proper intermediate in the cholesterol side-chain cleavage reaction. In addition, among various steroid complexes of ferrous-NO species having rhombic symmetry, the EPR spectra of 20(R),22(R)-dihydroxycholesterol complexes were significantly different from those of 22(R)-hydroxycholesterol complexes, suggesting that upon 20S-hydroxylation of 22(R)-hydroxycholesterol the conformation of the active site changes so as to facilitate subsequent cleavage of the C20-C22 bond of the cholesterol side chain. Addition of reduced adrenodoxin to the ferrous-NO cytochrome P-450scc complex in the presence of cholesterol caused a complete shift of the gx = 2.070 signal to gx = 2.075, indicating a reorientation of cholesterol in the substrate-binding site of the enzyme upon adrenodoxin binding. Without reduced adrenodoxin, the process of reorientation of cholesterol in the substrate-binding site was very slow, requiring more than 50 h of incubation at 0 degrees C. The present observations suggest that adrenodoxin may have another positive role in the cholesterol side-chain cleavage reaction, in addition to transferring an electron to the heme of cytochrome P-450scc.
Tsubaki
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Hiwatashi
A
A
Ichikawa
Y
Y
Fujimoto
Y
Y
Ikekawa
N
N
Hori
H
H
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation
D006888:Hydroxycholesterols; D000325:Adrenodoxin; D009569:Nitric Oxide; C003563:20,22-dihydroxycholesterol; D002786:Cholesterol Side-Chain Cleavage Enzyme