NMR-STUDIES OF SUBSTRATE-BINDING TO CYTOCHROME-P-450 (BM3) - COMPARISONS TO CYTOCHROME-P-450 (CAM)
NMR-STUDIES OF SUBSTRATE-BINDING TO CYTOCHROME-P-450 (BM3) - COMPARISONS TO CYTOCHROME-P-450 (CAM)
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DOI：
10.1021/bi00028a006
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发表时间：
1995-07-18
期刊：
影响因子：
2.9
通讯作者：
ROBERTS, GCK
中科院分区：
文献类型：
--
作者：
MODI, S;PRIMROSE, WU;ROBERTS, GCK
本文研究了底物月桂酸钠和12-溴月桂酸钠与巨芽孢杆菌细胞色素P-450 (BM3) （CYP102）含血红素结构域的结合，方法是测量血红素铁的未配对电子对水和结合底物质子的弛豫效应。底物结合导致血红素铁从低自旋状态转化为高自旋状态，如光谱变化所示。弛豫测量表明，这伴随着铁的第六个配位上的水被排出，铁和水质子之间的距离从2.6埃增加到5.2埃。对底物质子进行相应的弛豫测量，可以确定结合底物的铁和质子之间的若干距离，从而获得底物在结合位点的位置和方向的信息。月桂酸盐和12-溴月桂酸盐的结合方式非常相似，羧酸盐可能靠近Arg47，而链的另一端距离血红素铁7.6-7.8埃。结果表明，月桂酸盐和吡啶可以同时结合到P-450结构域上，该三元配合物中铁-月桂酸盐的距离与二元配合物中的距离没有显著差异。这些观察结果与细胞色素P-450 （cam）的底物复合物的观察结果进行了比较，并讨论了它们对催化循环中涉及的结构变化的影响。
The binding of the substrates sodium laurate and sodium 12-bromolaurate to the heme-containing domain of Bacillus megaterium cytochrome P-450 (BM3) (CYP102) has been studied by measurement of the relaxation effects of the unpaired electrons of the heme iron on the protons of water and of the bound substrates. Substrate binding leads to a conversion of the heme iron from a low-spin to a high-spin state, as shown by changes in the optical spectrum. The relaxation measurements show that this is accompanied by expulsion of water from the sixth coordination position of the iron, the distance between the iron and the water protons increasing from 2.6 to 5.2 Angstrom. Corresponding relaxation measurements on the substrate protons lead to the determination of a number of distances between the iron and protons of the bound substrate and, hence, to information on the position and orientation of the substrate in the binding site. Laurate and 12-bromolaurate are found to bind in a very similar way, in an extended conformation with the carboxylate probably close to Arg47 and the other end of the chain 7.6-7.8 Angstrom from the heme iron. It is shown that laurate and pyridine can bind simultaneously to the P-450 domain and that the iron-laurate distances in this ternary complex are not significantly different from those in the binary complex. These observations are compared with those on the substrate complex of cytochrome P-450 (cam), and their implications for structural changes involved in the catalytic cycle are discussed.