Conformational and functional studies of chemically modified cytochrome c: nitrated and iodinated cytochromes c.
Conformational and functional studies of chemically modified cytochrome c: nitrated and iodinated cytochromes c.
复制标题
化学修饰细胞色素 c 的构象和功能研究：硝化和碘化细胞色素 c。
DOI：
10.1021/bi00690a029
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
Y. Myer
中科院分区：
文献类型：
--
作者：
P. K. Pal;B. Verma;Y. Myer
碘化（E. B。McGowan和E. Stellwagen（1970），Biochemistry 9，3074）和硝化的（M. Sokolovsky等人（1970），Biochemistry 9，5113）细胞色素c导致从具有Tyr-67和Tyr-74的修饰的二碘代双酪氨酸基-细胞色素c（DIDT-）的前一种制备物中回收，并且从后者中回收具有Tyr-67的修饰的单硝基单酪氨酸基-细胞色素c（MNMT-）和具有添加的Tyr-48的修饰的单硝基双酪氨酸基-细胞色素c（MNDT-）。三个纯化的制剂进行了构象，其特征在于使用pH-光谱，圆二色性，热变性，还原与抗坏血酸，自氧化与分子氧，并与CO结合。这些结果是相关的两个方面的生物功能，还原，测量由NADH-细胞色素c还原酶，和氧化能力，与细胞色素c氧化酶，以及在蛋白质的结构-功能关系。MNMT-细胞色素c在结构上和构象上都是一种单一的异构体，可被抗坏血酸还原，对分子氧的氧化和CO的结合都具有小但确定的亲和力。在构象上，在金属原子的两种价态下，它代表一种具有天然构象的分子形式，在血红素基团附近具有小但确定的扰动，这反映在Met-80-S-Fe键的不稳定。MNMT-铁细胞色素c表现出6.2的pK，用于将含有695 nm带的低自旋、天然样光谱形式II转化为缺乏695 nm带的形式。当根据pK为9.2的天然蛋白质的异构化和所涉及基团的性质进行分析时，在pK = 6.2处的异构化表明Tyr-67不参与修饰制剂的异构化，并且可能也不参与天然蛋白质。在生物学功能方面，可重新识别性的部分紊乱（24%）和未改变的氧化性指向Tyr-67的功能意义，并提供了生理功能功能两个方面之间的选择性的另一个例子，与蛋白质的双功能，双路径操作模型一致。的MNDT-和DIDT-ferricytochromes C表现出的物理化学性质指示的蛋白质的构象以及金属原子的配位构型的总的紊乱。在这两种情况下，完全不能接受来自NADH-细胞色素c还原酶的电子，并且保留了50%的DIDT-细胞色素c的氧化性，被解释为构象紊乱的结果，而不是Tyr-48或Tyr-74的附加修饰。
The purification of iodinated (E. B. McGowan and E. Stellwagen (1970), Biochemistry 9, 3074) and of nitrated (M. Sokolovsky et al. (1970), Biochemistry 9, 5113) cytochromes c resulted in the recovery from the former preparation of diiododityrosyl-cytochrome c (DIDT-) with modification of Tyr-67 and Tyr-74, and, from the latter, a mononitromonotyrosyl-cytochrome c (MNMT-), with modification of Tyr-67, and mononitrodityrosyl-cytochrome c (MNDT-), with the added modification of Tyr-48. The three purified preparations were conformationally characterized using pH-spectroscopy, circular dichroism, thermal denaturation, reducibility with ascorbate, autoxidation with molecular oxygen, and binding with CO. These results are related to the two aspects of biological function, reducibility, measured by NADH-cytochrome c reductase, and oxidizability, with cytochrome c oxidase, as well as to structure-function relationships in the protein. MNMT-cytochrome c was found to be, structurally and conformationally, a single isomer, reducible with ascorbate, with a small, but definite affinity for both oxidation with molecular oxygen and binding of CO. Conformationally, in both valence states of the metal atom, it represents a molecular form with native-like conformation with small but definite perturbations in the immediate vicinity of the heme group, reflected by the destabilization of the Met-80-S-Fe linkage. MNMT-ferricytochrome c exhibits a pK of 6.2 for the transformation of the low-spin, native-like spectral form II containing the 695-nm band to form lacking lacking the 695-nm band. The isomerization at pK = 6.2, when analyzed in terms of the isomerization of the native protein with a pK of 9.2 and the nature of the group involved, indicates that Tyr-67 is not involved in the isomerization of the modified preparation, and possibly not in the native protein as well. In terms of biological function, the partial derangement of redecibility (24%) and the unaltered oxidizability point to the functional significance of Tyr-67, and provide another example of selectivity between the two aspects of physiological functional function, in agreement with the two-function, two-path operational model of the protein. The MNDT- and DIDT-ferricytochromes c exhibited physicochemical properties indicative of gross derangement of both the conformation of the protein as well as of the coordination configuration of the metal atom. The complete inability to accept an electron from NADH-cytochrome c reductase in both cases, and the retention of 50% of the oxidizability property of DIDT-cytochrome c, were interpreted to be the result of conformational derangement, rather than the added modification of Tyr-48 or of Tyr-74.