The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase
The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase
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DOI：
10.1046/j.1432-1327.1999.00249.x
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发表时间：
1999-04-01
期刊：
影响因子：
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通讯作者：
Soulimane, T
中科院分区：
文献类型：
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作者：
Döpner, S;Hildebrandt, P;Soulimane, T
酵母iso-1细胞色素c与牛细胞色素c氧化酶的相互作用进行了研究，使用细胞色素c的变体，其中结合结构域的赖氨酸被丙氨酸取代。两种蛋白质的完全氧化复合物的共振拉曼光谱揭示了血红素c以及血红素a和a的结构变化（3）。细胞色素c的结构变化与结合磷脂囊泡时观察到的相同，其中结合蛋白存在于两种构象异构体B1和B2中。虽然B1的结构与未结合的细胞色素c的结构相同，但B2的形成与血红素口袋的实质性改变有关。在细胞色素c氧化酶中，两种血红素的结构变化是指直接蛋白质环境的更微妙的扰动，可能是涉及两种状态的构象平衡的结果。这些变化是定性不同的细胞色素c氧化酶后，聚-L-赖氨酸的结合。定量分析了各种细胞色素c/细胞色素c氧化酶复合物的共振拉曼光谱。通过对相同蛋白质组合的稳态动力学测量来验证光谱研究。的光谱分析和动力学研究的结果被用来确定的复合物的稳定性和构象平衡B2/B1的所有细胞色素c的变体。复合物稳定性按以下顺序降低：野生型WT > J72 K> K79 A> K73 A> K87 A> J72 A> K86 A> K73 A/K79 A（其中J是天然三甲基赖氨酸）。构象平衡不显示这种顺序。状态B2形成的静电控制不依赖于单独的分子间盐桥，而是依赖于细胞色素c前表面的特定区域中的电荷分布，该区域由位置72、73和79处的赖氨酰残基限定。另一方面，在细胞色素c氧化酶的构象变化被发现是独立的绑定细胞色素c的变体的身份。从稳态动力学测量确定的最大速率常数可能与使用一个简单的模型，假设构象转变比产品形成更快的结合细胞色素c的构象平衡。在该模型中，数据分析得出的结论是，状态B2中的蛋白质间电子转移速率常数比B1中高约两倍。这些结果可以解释为，由于还原电位的大的负偏移，状态B2中的驱动力增加。
The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a(3). The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to these observed for cytochrome c oxidase upon poly-L-lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.