Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme.
Spectroscopic investigations of ferric cytochrome P-450-CAM ligand complexes. Identification of the ligand trans to cysteinate in the native enzyme.
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DOI：
10.1016/s0021-9258(18)34823-3
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发表时间：
1982-04
期刊：
影响因子：
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通讯作者：
John H. Dawson;Laura A. AnderssonS;M. Sono
中科院分区：
文献类型：
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作者：
John H. Dawson;Laura A. AnderssonS;M. Sono
通过紫外-可见吸收、磁圆二色性和电子顺磁共振光谱检查了一系列广泛的三价细胞色素 P-450-CAM 配体复合物，试图在酶的六配位静止状态下鉴定反式半胱氨酸的配体。因此，所使用的配体被选择作为潜在内源氨基酸配位的模型，包括醇、酰胺和羧酸盐氧供体、胺、咪唑和吲哚氮供体以及二硫化物、硫醚、硫醇和硫醇盐硫供体。由于这项研究本质上是一项实证研究，因此通过同时使用三种不同的光谱技术来加强结论。除硫醇盐加成产生的络合物外，所有形成的络合物都显示出与低自旋、六配位 P-450 大致相似的光谱特性。在硫供体加合物中，二硫化物和硫醚结合的 P-450 具有足够细节的不同特性，足以将它们与天然 P-450 区分开来。虽然硫醇结合物质和低自旋三价铁 P-450 的光谱特征相似，但前者由于与结合硫醇盐的相互转化而具有 pH 依赖性，而后者基本上不随 pH 值而变化。在氧供体复合物中，除羧酸盐外，所有复合物的光谱都与静息酶的光谱非常匹配。与大多数含氮配体（包括几种咪唑衍生物）形成的加合物表现出的光谱与天然 P-450 有很大不同，因此将它们排除在第六种配体的候选者之外。有趣的是，与在α位具有大吸电子取代基的咪唑衍生物形成的络合物的光谱性质与那些天然P-450相当，除了EPR光谱的线形之外。先前发表的理论工作表明，这种咪唑衍生物与其他检查的衍生物之间的光谱差异是电子性的，而不是空间性的。由于该蛋白质不存在类似的电子机制来降低给电子能力或组氨酸，因此认为可以排除组氨酸在P-450的第六位上的配位。总之，对所有光谱数据的仔细检查表明，P-450-CAM 与酰胺（特别是醇）的氨基酸类似物加合物产生的光谱几乎完全复制天然 P-450 的光谱，并表明六配位铁酶中反式至半胱氨酸的配体具有氧供体原子。
An extensive series of ligand complexes of ferric cytochrome P-450-CAM has been examined by UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy in an attempt to identify the ligand trans to cysteinate in the six-coordinate resting state of the enzyme. Thus, the ligands used have been chosen to serve as models for coordination by potential endogenous amino acids and include alcohol, amide and carboxylate oxygen donors, amine, imidazole and indole nitrogen donors and disulfide, thioether, thiol, and thiolate sulfur donors. As this investigation has been by nature an empirical one, the conclusions are strengthened by the concurrent use of three different spectroscopic techniques. All of the complexes formed except those resulting from thiolate addition display spectroscopic properties that are broadly similar to those of low spin, six-coordinate P-450. Of the sulfur donor adducts, disulfide and thioether-bound P-450 have properties that are different enough in detail to distinguish them from native P-450. While the spectral features of the thiol-bound species and of low spin ferric P-450 are alike, the former are pH dependent due to interconversion to bound thiolate, whereas the latter display essentially no spectral changes with pH. Of the oxygen donor complexes, all but carboxylate have spectra that very closely match those of the resting enzyme. Adducts formed with most nitrogenous ligands, including several imidazole derivatives, exhibit spectra that are sufficiently different from native P-450 to exclude them as candidates for the sixth ligand. Interestingly, the spectral properties of a complex formed with an imidazole derivative having a bulky electron-withdrawing substituent in the alpha position are comparable to those native P-450 except for the line shape of the EPR spectrum. Previously published theoretical work suggests that the spectral differences seen between this imidazole derivative and the other examined are electronic and not steric in origin. As no similar electronic mechanism exists for the protein to reduce the electron-donating ability or histidine, it is felt that coordination of histidine in the sixth position of P-450 can be ruled out. In conclusion, close examination of all spectral data reveals that amino acid analog adducts of P-450-CAM with amides and, in particular, alcohols, produce spectra that almost exactly duplicate those of native P-450 and suggests that the ligand trans to cysteinate in the six-coordinate ferric enzyme has an oxygen donor atom.