Elmer-Dixon Margaret M, Bowler Bruce E
Department of Chemistry and Biochemistry, University of Montana , Missoula, Montana 59812, United States.
Center for Bimolecular Structure and Dynamics, University of Montana , Missoula, Montana 59812, United States.
Biochemistry. 2017 Sep 12;56(36):4830-4839. doi: 10.1021/acs.biochem.7b00694. Epub 2017 Aug 30.
Measurements at pH 8 allow evaluation of binding of 100% cardiolipin vesicles to site A of cytochrome c without interference from other known binding sites. Site A encompasses Lys72, Lys73, Lys86, and Lys87, located in or adjacent to Ω-loop D (residues 70-85), which positions Met80 for binding to the heme. Binding of cytochrome c to cardiolipin disrupts Met80 heme binding, permitting peroxidase activity. Binding of cardiolipin to yeast iso-1-cytochrome c versus human cytochrome c is compared to assess how binding of cardiolipin to site A has evolved for cytochrome c from species that do not have a complete intrinsic apoptotic pathway to species that do. Using a nondestructive method of quantifying cardiolipin concentration, highly reproducible binding curves are obtained. The results indicate two sequential structural rearrangements on the surface of 100% cardiolipin vesicles. The first, more modest, structural rearrangement occurs at an exposed (outer leaflet) lipid:protein ratio of 8-10 for both cytochromes c. The second, occurring at higher lipid:protein ratios, causes significant unfolding of cytochrome c and requires a much higher lipid:protein ratio for human versus yeast cytochrome c. Higher lipid:protein ratios enhance the peroxidase activity of cytochrome c, suggesting that human cytochrome c has evolved a more stringent on/off switch for cardiolipin peroxidation in the early stages of apoptosis. For both human and yeast cytochrome c, the K72A mutation has only minor effects on binding to site A, suggesting that other nearby lysines can compensate for the lack of Lys72.
在pH 8条件下进行测量，可以评估100%的心磷脂囊泡与细胞色素c的A位点的结合情况，而不会受到其他已知结合位点的干扰。A位点包括位于Ω-环D（残基70 - 85）内或其附近的赖氨酸72、赖氨酸73、赖氨酸86和赖氨酸87，该环将甲硫氨酸80定位以与血红素结合。细胞色素c与心磷脂的结合会破坏甲硫氨酸80与血红素的结合，从而使过氧化物酶活性得以发挥。比较心磷脂与酵母同工酶-1-细胞色素c和人细胞色素c的结合情况，以评估从没有完整内在凋亡途径的物种到具有完整内在凋亡途径的物种，细胞色素c的心磷脂A位点结合是如何演变的。使用一种无损定量心磷脂浓度的方法，可以获得高度可重复的结合曲线。结果表明在100%的心磷脂囊泡表面发生了两个连续的结构重排。第一个结构重排较为温和，对于两种细胞色素c而言，在暴露的（外层小叶）脂质与蛋白质比例为8 - 10时发生。第二个结构重排发生在更高的脂质与蛋白质比例下，会导致细胞色素c显著展开，并且人细胞色素c所需的脂质与蛋白质比例比酵母细胞色素c高得多。更高的脂质与蛋白质比例会增强细胞色素c的过氧化物酶活性，这表明人细胞色素c在凋亡早期阶段已经进化出了一种更严格的心磷脂过氧化开启/关闭开关。对于人和酵母细胞色素c来说，K72A突变对与A位点的结合只有轻微影响，这表明附近的其他赖氨酸可以补偿赖氨酸72的缺失。
