Lin J, Wu S, Chan S I
A. A. Noyes Laboratory of Chemical Physics, California Institute of Technology, Pasadena 91125, USA.
Biochemistry. 1995 May 16;34(19):6335-43. doi: 10.1021/bi00019a011.
Bovine heart cytochrome c oxidase (CcO) has been modified by 8-azido-adenosine 5'-triphosphate (8-azido-ATP), and the electron-transfer activity from ferrocytochrome c to the modified CcO under physiological ionic strengths has been studied by the laser flash photolysis technique with 5-deazariboflavin and EDTA as the electron donor. The kinetics of intermolecular electron transfer between the redox protein partners was shown to be reduced significantly. In addition, there is significant decrease in the binding affinity of the cytochrome c to the oxidase upon 8-azido-ATP modification. The 8-azido-ATP-modified CcO exhibited 50% of the intracomplex electron-transfer rate (ket) and 56% of the association constant (Ka) normally observed between cytochrome c and native CcO under otherwise identical conditions. Since the effective electron transfer rate constant is the product of ket and Ka under nonsaturation conditions, the overall electron-transfer rate has been curtailed by over a factor of 2. Similar observations have been noted with the native CcO in the presence of 3 mM ATP. In contrast, the redox potential of neither CuA nor cytochrome a was altered upon 8-azido-ATP modification or in the presence of 3 mM ATP. Also, no gross structural changes at either the CuA or the cytochrome a site were noted, as evidenced by a lack of any spectral perturbations in the EPR signals from both of these centers. We conclude that ATP modulates the electron transfer from cytochrome c to CcO by interacting with the CcO and altering allosterically the docking. In this manner, ATP can affect the branching of the electron input from ferrocytochrome c to cytochrome a and CuA.
牛心细胞色素c氧化酶（CcO）已被8-叠氮腺苷5'-三磷酸（8-叠氮-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从亚铁细胞色素c到修饰后的CcO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动力学显著降低。此外，8-叠氮-ATP修饰后，细胞色素c与氧化酶的结合亲和力显著降低。在其他条件相同的情况下，8-叠氮-ATP修饰的CcO表现出细胞色素c与天然CcO之间通常观察到的复合物内电子转移速率（ket）的50%和缔合常数（Ka）的56%。由于在非饱和条件下有效电子转移速率常数是ket和Ka的乘积，因此整体电子转移速率已降低了两倍多。在存在3 mM ATP的情况下，天然CcO也有类似的观察结果。相比之下，8-叠氮-ATP修饰或存在3 mM ATP时，CuA和细胞色素a的氧化还原电位均未改变。此外，CuA或细胞色素a位点均未观察到明显的结构变化，这两个中心的电子顺磁共振信号中没有任何光谱扰动证明了这一点。我们得出结论，ATP通过与CcO相互作用并变构改变对接来调节从细胞色素c到CcO的电子转移。通过这种方式，ATP可以影响从亚铁细胞色素c到细胞色素a和CuA的电子输入分支。
