Nicholls P, Hildebrandt V, Hill B C, Nicholls F, Wrigglesworth J M
Can J Biochem. 1980 Oct;58(10):969-77. doi: 10.1139/o80-132.
In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.
在低离子强度介质中，膜结合细胞色素c氧化酶、分离的细胞色素c氧化酶和蛋白脂质体细胞色素c氧化酶各自在两个位点结合细胞色素c，一个是低亲和力位点（1微摩尔大于解离常数Kd'大于0.2微摩尔）且易于可逆结合，另一个是高亲和力位点（0.01微摩尔大于Kd）且结合较弱可逆。当细胞色素c占据两个位点，包括低亲和力位点时，用抗坏血酸和N，N，N'，N'-四甲基对苯二胺（TMPD）进行极谱法测量的最大周转率与TMPD浓度无关，对于完全活化的系统，在30℃、pH 7.4时，其值在250至400 s-1之间。然而，该酶对细胞色素c的表观亲和力取决于TMPD。当细胞色素c仅占据高亲和力位点时，最大周转率紧密依赖于TMPD的浓度，与抗坏血酸不同，TMPD可以还原结合的细胞色素c。随着TMPD浓度增加，最大周转率接近两个位点都被占据时的情况。分离的细胞色素aa3活性较低是由于存在无活性或无法接近的酶分子。将分离的酶掺入磷脂囊泡可使所有随后可接近的细胞色素aa3分子恢复完全活性。带负电荷的（大豆卵磷脂）囊泡在低亲和力位点显示出比其他酶制剂更高的细胞色素c亲和力。提出了细胞色素c - 细胞色素aa3复合物的模型，其中两个位点被占据时都具有完全催化活性，但通过“松散”位点对细胞色素c进行整体氧化需要一个具有催化功能的细胞色素c分子占据“紧密”位点。
