Lin J, Wu S, Lau W T, Chan S I
A.A. Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125.
Biochemistry. 1995 Feb 28;34(8):2678-85. doi: 10.1021/bi00008a035.
Horse heart cytochrome c has been modified by 8-azido-ATP and the electron-transfer activity of the modified cytochrome c's to bovine heart cytochrome c oxidase (CcO) under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The intermolecular electron transfer between the redox protein partners was shown to be extremely slow. The 8-azido-ATP-modified system exhibited less than 5% of the intracomplex electron-transfer rate observed between native cytochrome c and CcO under otherwise identical conditions. The binding affinity of the modified cytochrome c was greatly reduced (3 orders of magnitude) at low ionic strengths; however, it was only slightly reduced (by a factor of 2) relative to the native protein at physiological ionic strengths. Thus, the binding affinity of the ATP-cytochrome c adducts is relatively insensitive to the ionic strength compared to the native enzyme, suggesting that a different docking conformation is assumed by the ATP-cytochrome c adducts in their interaction with the oxidase. Since the redox potential of the modified cytochrome c is close to the value of its native form, we conclude that there has been a change in the docking of the cytochrome c to CcO and the electronic coupling between heme c and CuA upon 8-azido-ATP modification.
马心脏细胞色素c已被8-叠氮基-ATP修饰，并且在生理离子强度下，以5-脱氮核黄素和EDTA作为电子供体，通过激光闪光光解技术研究了修饰后的细胞色素c与牛心脏细胞色素c氧化酶（CcO）之间的电子转移活性。结果表明，氧化还原蛋白伴侣之间的分子间电子转移极其缓慢。在其他条件相同的情况下，8-叠氮基-ATP修饰的体系表现出的复合体内电子转移速率不到天然细胞色素c与CcO之间所观察到的5%。在低离子强度下，修饰后的细胞色素c的结合亲和力大幅降低（3个数量级）；然而，在生理离子强度下，相对于天然蛋白，其结合亲和力仅略有降低（降低了2倍）。因此，与天然酶相比，ATP-细胞色素c加合物的结合亲和力对离子强度相对不敏感，这表明ATP-细胞色素c加合物在与氧化酶相互作用时采取了不同的对接构象。由于修饰后的细胞色素c的氧化还原电位接近其天然形式的值，我们得出结论，在8-叠氮基-ATP修饰后，细胞色素c与CcO的对接以及血红素c与CuA之间的电子耦合发生了变化。
