Tsubaki M, Ichikawa Y, Fujimoto Y, Yu N T, Hori H
Department of Biochemistry, Kagawa Medical School, Japan.
Biochemistry. 1990 Sep 18;29(37):8805-12. doi: 10.1021/bi00489a043.
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)
细胞色素P - 45011β从牛肾上腺皮质线粒体中以与11 - 脱氧皮质酮结合的形式被纯化出来，并通过共振拉曼光谱和电子顺磁共振光谱对其活性位点进行了研究。纯化样品的共振拉曼光谱显示，基于ν10（1629cm-1）和ν3（1490 cm-1）模式频率，血红素铁采用纯五配位铁高自旋态，这些频率高于六配位铁高自旋细胞色素P - 450scc - 底物复合物的频率。在亚铁 - CO状态下，基于同位素取代技术，在481.5 cm-1处鉴定出一个Fe2(+) - CO伸缩模式；该频率与胆固醇复合状态下的细胞色素P - 450scc的频率（483 cm-1）非常接近。纯化样品在4.2 K时的电子顺磁共振光谱显示出铁高自旋信号（g = 7.98、3.65和1.71），这与细胞色素P - 450scc的铁高自旋信号（g = 8.06、3.55和1.68）明显不同，并证实了肾上腺皮质线粒体中铁高自旋信号的先前归属。亚铁细胞色素P - 45011β的一氧化氮（NO）复合物的电子顺磁共振光谱显示出具有菱形对称性的电子顺磁共振信号（gx = 2.068，gz = 2.001，gy = 1.961），与在77 K下存在22(S) - 羟基胆固醇和20(R),22 - (R) - 二羟基胆固醇时亚铁细胞色素P - 450scc - NO复合物的信号非常相似。（摘要截断于250字）
