Chemical modification of the CuA center in cytochrome c oxidase by sodium p-(hydroxymercuri)benzoate.
Chemical modification of the CuA center in cytochrome c oxidase by sodium p-(hydroxymercuri)benzoate.
复制标题
对（羟基汞）苯甲酸钠对细胞色素 C 氧化酶中 CuA 中心的化学修饰。
DOI：
10.1021/bi00336a025
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Chan,SI
中科院分区：
文献类型：
--
作者：
Gelles,J;Chan,SI
细胞色素C氧化酶包含一个铜离子电子转移位点CUA，以前发现在蛋白质保持结构完整的条件下，该位点不与外部添加的试剂发生反应。我们研究了细胞色素氧化酶与对羟基汞苯甲酸钠(PHMB)的反应，发现在适当的条件下，该反应得到了一种特殊的CuA中心的衍生物，其电子顺磁共振和近红外吸收光谱性质与未经修饰的中心明显不同。对酶的其他金属离子位置的光谱和化学表征表明，PHMB修饰对这些位置的结构和反应几乎没有影响。特别令人感兴趣的是，观察到修饰的酶仍然显示出从细胞色素c铁到02的电子转移的天然稳态活性的相当大一部分。虽然修饰的铜中心保留了从强大的还原剂Na2S204接收电子并将电子转移到02的能力，但当用较温和(较高电位)的还原剂如NADH/吩嗪甲硫酸盐或生理底物铁细胞色素c处理酶时，它不会显著还原。CUA显示出许多光谱和化学性质，使其高度非典型的CuProtein活性中心；这个位置的奇异性质促使人们猜测这种金属离子中心的结构特性在酶的催化循环中的重要性。在这项工作中，我们证明了这个位点的不同寻常的特征不是有效催化电子转移和酶还原O2的先决条件。具体地说，这些观察结果支持这样的模型，即细胞色素c和氧结合部位之间存在不涉及CUA的第二次电子转移途径，氧结合部位的作用速度至少是天然酶的20%。(细胞色素c氧化酶催化线粒体电子传递链的最后一步，在该步骤中，细胞色素c铁氧化产生的电子在氧还原为水的过程中消耗。该酶的每个分子含有两个铜离子和两个血红素。这四个金属中心中有三个已知在催化循环中发挥关键作用：其中一个血红素(称为Fea)*是细胞色素c的主要电子受体，它在酶中的位置被认为接近细胞色素c结合部位(Malmstrom，1980)[尽管另一种观点见Capaldi等人(1983)]；另一个血红素(Feaj)和其中一个铜离子(Cub)一起组成双核部位，结合02和02衍生的中间体，同时它们经历复杂的化学转化序列，最终导致两个H20分子的合成。02结合位点被认为存在于蛋白质内部的疏水环境中(Fiamingo等人，1982；Alben等人，1981)，并且距离FeA很远(约20？)(Brudvig等人，1984)。因此，酶必须包含连接两个位点的电子转移途径，并促进电子在周转过程中的快速移动。
Cytochrome c oxidase contains a copper ion electron-transfer site, CuA, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-(hydroxymercuri) benzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the CuA center that has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to 02. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na2S204 and to transfer electrons to 02, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c. CuA exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this sitehas prompted speculation about the importance of the structural peculiarities of this metal ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and 02 reduction by the enzyme. Specifically, these observations support the modelin which a secondary electron-transfer pathway not involving CuA exists between the cytochrome c and oxygen binding sites which can function at a rate at least 20% of thatin the native enzyme.(yytochrome c oxidase catalyzes the final step of the mitochondrial electron transport chain in which electrons derived from the oxidation of ferrocytochrome c are consumed in the reduction of oxygen to water. Each molecule of the enzyme contains two copper ions and two hemes. Three of these four metal centers are known to play critical roles in the catalytic cycle: one of the hemes (referred to as Fea)* is the primary electron acceptor from cytochrome c, and its location within the enzyme is presumed to be near that of the cytochrome c binding site (Malmstrom, 1980)[although for an alternative view see Capaldi et al.(1983)]; the otherheme (Feaj) and one of the copper ions (CuB) together make up the binuclear site that binds 02 and 02-derived intermediates while they undergo a complex sequence of chemical conversions thatultimately result in the synthesis of two H20 molecules. The02-binding site is thought to reside in a hydrophobic environment inside the protein (Fiamingo et al., 1982; Alben et al., 1981), and is located a substantial distance (approximately 20 Á) away from Fea (Brudvig et al., 1984). The enzyme must therefore contain an electron-transfer pathway that connects the two sites and facilitates the rapid movement of electrons during turnover.