Hennig B, Koehler H, Neupert W
Proc Natl Acad Sci U S A. 1983 Aug;80(16):4963-7. doi: 10.1073/pnas.80.16.4963.
Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria.
细胞色素c的组装涉及一系列步骤：在游离核糖体上合成脱辅基细胞色素c，脱辅基细胞色素c与线粒体表面特异性结合，穿过外膜转移，原血红素的共价添加，多肽链的重折叠，以及全细胞色素c与其在内膜上的功能位点的缔合。通过用血红素类似物去铁血红素抑制后续转移步骤，研究了脱辅基细胞色素c与粗糙脉孢菌线粒体的结合步骤。结合位点对线粒体脱辅基细胞色素c具有高度特异性。结合的标记粗糙脉孢菌脱辅基细胞色素c被来自各种物种的未标记脱辅基细胞色素c竞争性取代。这些物种表现出不同的取代能力。反硝化副球菌的脱辅基细胞色素c、粗糙脉孢菌脱辅基细胞色素c的氨基末端（血红素结合）片段和粗糙脉孢菌全细胞色素c不能识别结合位点。聚赖氨酸不干扰脱辅基细胞色素c的结合。脱辅基细胞色素c是可逆结合的。结合位点数量有限。高亲和力结合位点的存在量约为90 pmol/mg线粒体蛋白。它们的缔合常数为2.2×10⁷ M⁻¹。当去铁血红素的抑制作用解除时，脱辅基细胞色素c被导入线粒体并直接从结合位点转化为全细胞色素c。我们得出结论，线粒体上的脱辅基细胞色素c结合位点代表了线粒体识别和导入这种前体的受体。
