Craig D B, Wallace C J
Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.
Biochemistry. 1995 Feb 28;34(8):2686-93. doi: 10.1021/bi00008a036.
We have proposed that the binding of ATP at a site of substantial affinity and specificity could regulate the activity of cytochrome c with its physiological partners and thus the overall efficiency of mitochondrial electron transport. We now describe the use of ATP affinity-labeled protein to test the effect of occupancy of that site, which includes the invariant arginine 91, on the activity of cytochrome c with purified cytochrome c reductase and oxidase and its association with the mitochondrial inner membrane. Electron-transfer activities with the reductase and oxidase were inhibited by site occupancy to 41% and 11-15% of native values, respectively. The marked difference in the degree of inhibition of activity that distinguishes the reactions with the two major physiological partners was sufficient to cause, in whole mitochondria, a demonstrable shift from a situation in which there is a rate-limiting transfer from the reductase to cytochrome c, to a state where rates are more evenly matched for transfers between cytochrome c and the two redox partners. Site occupancy also substantially reduces the ionic strength necessary for half-maximal dissociation of cytochrome c from the membrane. These data imply that the decreased efficiency of electron transfer caused by ATP attachment can be attributed to a decrease in the protein's activity with individual physiological partners, possibly compounded with a decrease in its affinity for the inner mitochondrial membrane, and suggest that feedback regulation by ATP of cellular respiration operates in like manner.
我们曾提出，ATP在具有高亲和力和特异性的位点上结合，可能会调节细胞色素c与其生理伴侣的活性，进而影响线粒体电子传递的整体效率。我们现在描述使用ATP亲和标记蛋白来测试该位点（包括不变的精氨酸91）被占据后，对细胞色素c与纯化的细胞色素c还原酶和氧化酶的活性及其与线粒体内膜结合的影响。位点被占据后，与还原酶和氧化酶的电子传递活性分别被抑制至天然值的41%和11 - 15%。与两种主要生理伴侣反应时活性抑制程度的显著差异，足以在完整线粒体中导致一种明显的转变，即从还原酶到细胞色素c存在限速转移的情况，转变为细胞色素c与两个氧化还原伴侣之间的转移速率更均匀匹配的状态。位点被占据还显著降低了细胞色素c从膜上半最大解离所需的离子强度。这些数据表明，ATP附着导致的电子传递效率降低，可归因于该蛋白与单个生理伴侣活性的下降，可能还伴有其对线粒体内膜亲和力的降低，并表明ATP对细胞呼吸的反馈调节以类似方式起作用。
