Tsubaki M, Hiwatashi A, Ichikawa Y, Fujimoto Y, Ikekawa N, Hori H
Department of Biochemistry, Kagawa Medical School, Japan.
Biochemistry. 1988 Jun 28;27(13):4856-62. doi: 10.1021/bi00413a041.
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.
在77K下，使用X波段（9.35GHz）微波频率测量了与20（R），22（R）-二羟基胆固醇复合的亚铁一氧化氮（14NO和15NO）细胞色素P-450scc的电子顺磁共振（EPR）光谱。EPR光谱清楚地表明自旋系统具有菱形对称性（对于14NO，gx = 2.068，gz = 2.001，gy = 1.961，Az = 1.89 mT），并且与20（S）-羟基胆固醇复合物的光谱不同。20（S）-羟基胆固醇复合物的独特性质表明20（S）-羟基胆固醇不是胆固醇侧链裂解反应中的合适中间体。此外，在具有菱形对称性的亚铁-NO物种的各种类固醇复合物中，20（R），22（R）-二羟基胆固醇复合物的EPR光谱与22（R）-羟基胆固醇复合物的光谱有显著差异，这表明在22（R）-羟基胆固醇的20S-羟基化后，活性位点的构象发生变化，从而促进胆固醇侧链C20-C22键的后续裂解。在胆固醇存在的情况下，向亚铁-NO细胞色素P-450scc复合物中加入还原型肾上腺皮质铁氧还蛋白会使gx = 2.070信号完全转变为gx = 2.075，这表明在肾上腺皮质铁氧还蛋白结合后，胆固醇在酶的底物结合位点发生了重新定向。没有还原型肾上腺皮质铁氧还蛋白时，胆固醇在底物结合位点的重新定向过程非常缓慢，在0℃下需要孵育超过50小时。目前的观察结果表明，肾上腺皮质铁氧还蛋白除了将电子转移到细胞色素P-450scc的血红素上之外，可能在胆固醇侧链裂解反应中还有另一个积极作用。
