PH-LINKED CONFORMATIONAL REGULATION OF A METALLOPROTEIN OXIDATION REDUCTION EQUILIBRIUM - ELECTROCHEMICAL ANALYSIS OF THE ALKALINE FORM OF CYTOCHROME-C
PH-LINKED CONFORMATIONAL REGULATION OF A METALLOPROTEIN OXIDATION REDUCTION EQUILIBRIUM - ELECTROCHEMICAL ANALYSIS OF THE ALKALINE FORM OF CYTOCHROME-C
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DOI：
10.1021/ja00036a006
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发表时间：
1992-05-06
影响因子：
15
通讯作者：
MAUK, AG
中科院分区：
文献类型：
--
作者：
BARKER, PD;MAUK, AG
通过循环伏安法，在宽范围的扫描速率和溶液 pH 范围内研究了热解石墨表面上各种细胞色素 c 的直接电化学，其中铁细胞色素 c 可以以天然构象或碱性构象存在。观察到的电化学与细胞色素 c 的两种构象异构体在该电极表面上完全电活性完全一致。扫描速率为 1 V s-1 及以上的实验表明，两种细胞色素构象异构体都表现为简单的单电子转移蛋白。这一发现首次测量了细胞色素 c 碱性形式的中点还原电位，从而可以对涉及蛋白质两种构象异构体的两种氧化态的碱性构象循环进行完整的热力学分析。该分析提供的证据的核心是从酵母 iso-1-细胞色素 c 的位点特异性变体获得的结果，这些变体改变了碱性 pK(a)。野生型酵母 iso-1-细胞色素 c 的碱性形式在 pH 8.45 下的还原电位相对于 SHE 为 -205 mV。马心蛋白在 pH 10.0 时的等效构象异构体也获得了相同的值。较低扫描速率下的实验与还原的碱性构象异构体到还原的天然构象异构体的重排一致，并且这种相互转化的速率是根据此处研究的所有细胞色素的扫描速率依赖性来估计的。这项研究强调了动态电化学分析在细胞色素 c 及其变体研究中的作用，这些变体经历了显着的氧化态相关构象变化。
The direct electrochemistry of a variety of cytochromes c at pyrolytic graphite surfaces has been studied by cyclic voltammetry over a wide range of sweep rate and over a range of solution pH in which ferricytochrome c can exist in either the native or alkaline conformations. The electrochemistry observed is entirely consistent with both conformers of cytochrome c being fully electroactive at this electrode surface. Experiments at sweep rates of 1 V s-1 and above show that both cytochrome conformers behave as simple one-electron-transfer proteins. This finding has provided the first measurement of the midpoint reduction potential for the alkaline form of cytochrome c and thereby allows a complete thermodynamic analysis of the alkaline conformational cycle involving both oxidation states of the two conformers of the protein. Central to the evidence provided for this analysis are the results obtained from site-specific variants of yeast iso-1-cytochrome c which have altered alkaline pK(a)s. The reduction potential of the alkaline form of wild-type yeast iso-1-cytochrome c at pH 8.45 is -205 mV vs SHE. The same value is obtained for the equivalent conformer of the horse heart protein at pH 10.0. Experiments at lower sweep rates are consistent with the rearrangement of the reduced alkaline conformer to the reduced native conformer, and the rates of this interconversion have been estimated from the sweep rate dependence for all the cytochromes studied here. This study highlights the role for dynamic electrochemical analysis in the study of cytochrome c and its variants which undergo significant oxidation state linked conformational changes.