Hall J, Zha X H, Yu L, Yu C A, Millett F
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Biochemistry. 1989 Mar 21;28(6):2568-71. doi: 10.1021/bi00432a033.
The reaction of Rhodobacter sphaeroides cytochrome c2 with the Rb. sphaeroides cytochrome bc1 complex was studied by using singly labeled cytochrome c2 derivatives. Cytochrome c2 was treated with chlorodinitrobenzoic acid to modify lysine amino groups to negatively charged carboxydinitrophenyllysines and separated into eight different fractions by ion-exchange chromatography on a Whatman SE 53 (sulfoxyethyl)cellulose column. Peptide mapping studies indicated that six of these fractions were modified at single lysine amino groups. Each of the derivatives had the same Vmax value as native cytochrome c2 in the steady-state reaction with the Rb. sphaeroides cytochrome bc1 complex. However, the Km values of the cytochrome c2 derivatives modified at lysines 10, 55, 95, 97, 99, and 106 were found to be larger than that of native cytochrome c2 by factors of 6, 2, 3, 32, 13, and 8, respectively. These results indicate that lysines located in the sequence 97-106 on the left side of the heme crevice have the greatest involvement in binding the cytochrome bc1 complex. The involvement of lysine 97 is especially significant because it is located in an extra loop comprising residues 89-98 that is not present in eukaryotic cytochrome c.
利用单标记细胞色素c2衍生物研究了球形红细菌细胞色素c2与球形红细菌细胞色素bc1复合物的反应。用氯代二硝基苯甲酸处理细胞色素c2，将赖氨酸氨基修饰为带负电荷的羧基二硝基苯基赖氨酸，并通过在Whatman SE 53（磺基氧乙基）纤维素柱上进行离子交换色谱分离成八个不同的组分。肽图谱研究表明，其中六个组分在单个赖氨酸氨基处被修饰。在与球形红细菌细胞色素bc1复合物的稳态反应中，每种衍生物的Vmax值与天然细胞色素c2相同。然而，发现赖氨酸10、55、95、97、99和106处修饰的细胞色素c2衍生物的Km值分别比天然细胞色素c2大6、2、3、32、13和8倍。这些结果表明，位于血红素裂隙左侧97-106序列中的赖氨酸在结合细胞色素bc1复合物中起最大作用。赖氨酸97的作用尤为显著，因为它位于真核细胞色素c中不存在的由89-98残基组成的额外环中。
