Hall J, Ayres M, Zha X H, O'Brien P, Durham B, Knaff D, Millett F
J Biol Chem. 1987 Aug 15;262(23):11046-51.
In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.
为了确定红螺菌细胞色素c2上与光合反应中心相互作用的结构域，将细胞色素c2上带正电荷的赖氨酸氨基进行修饰，形成带负电荷的羧基二硝基苯基赖氨酸。反应混合物通过在羧甲基纤维素和磺丙基 - 琼脂糖上的离子交换色谱法分离成六个不同的组分。肽图谱研究表明，组分A由细胞色素c2背面赖氨酸58、81和109处修饰的单标记衍生物混合物组成。发现组分C1、C2、C3和C4是细胞色素c2正面围绕血红素裂隙的赖氨酸9、13、75、86和88处修饰的单标记衍生物混合物。在用激光脉冲激发后，测量从红螺菌纯化的反应中心对羧基二硝基苯基 - 细胞色素c2衍生物的光氧化作用。发现背面赖氨酸处修饰的组分A的二级速率常数为2.3×10⁷ M⁻¹ s⁻¹，与天然细胞色素c2的2.6×10⁷ M⁻¹ s⁻¹几乎相同。然而，发现组分C1 - C4的速率常数比天然细胞色素c2的速率常数小6至12倍。这些结果表明，细胞色素c2血红素裂隙周围的赖氨酸参与了与反应中心结合位点处羧酸盐基团的静电相互作用。还测量了用三氟乙酰基或三氟甲基苯基氨基甲酰基修饰单个赖氨酸氨基的马心细胞色素c衍生物的反应速率。发现血红素裂隙周围的赖氨酸8、13、25、27、72、79或87的修饰显著降低了反应速率，而其他区域赖氨酸的修饰没有影响。这表明马心细胞色素c与反应中心的反应也涉及血红素裂隙结构域。
