Active site of bovine adrenocortical cytochrome P-450(11) beta studied by resonance Raman and electron paramagnetic resonance spectroscopies: distinction from cytochrome P-450scc.
Cytochrome P-45011 beta was purified as the 11-deoxycorticosterone-bound form from bovine adrenocortical mitochondria and its active site was investigated by resonance Raman and EPR spectroscopies. Resonance Raman spectra of the purified sample revealed that the heme iron adopts the pure pentacoordinated ferric high-spin state on the basis of the nu 10 (1629cm-1) and nu 3 (1490 cm-1) mode frequencies, which are higher than those of the hexacoordinated ferric high-spin cytochrome P-450scc-substrate complexes. In the ferrous-CO state, a Fe2(+)-CO stretching mode was identified at 481.5 cm-1 on the basis of an isotopic substitution technique; this frequency is very close to that of cytochrome P-450scc in the cholesterol-complexed state (483 cm-1). The EPR spectra of the purified sample at 4.2 K showed ferric high-spin signals (at g = 7.98, 3.65, and 1.71) that were clearly distinct from the cytochrome P-450scc ferric high-spin signals (g = 8.06, 3.55, and 1.68) and confirmed previous assignments of ferric high-spin signals in adrenocortical mitochondria. The EPR spectra of the nitric oxide (NO) complex of ferrous cytochrome P-45011 beta showed EPR signals with rhombic symmetry (gx = 2.068, gz = 2.001, and gy = 1.961) very similar to those of the ferrous cytochrome P-450scc-NO complex in the presence of 22(S)-hydroxycholesterol and 20(R),22-(R)-dihydroxycholesterol at 77 K.(ABSTRACT TRUNCATED AT 250 WORDS)
Tsubaki
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Ichikawa
Y
Y
Fujimoto
Y
Y
Yu
N T
NT
Hori
H
H
D000302:Adrenal Cortex / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013252:Steroid 11-beta-Hydroxylase / Q000737:chemistry*
D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme