Methionine-oxidized horse heart cytochromec. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage
Methionine-oxidized horse heart cytochromec. III. Ascorbate reduction and the methionine-80-sulfur-iron linkage
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蛋氨酸氧化的马心细胞色素。
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发表时间：
1989
期刊：
影响因子：
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通讯作者：
Swatantar Kumar
中科院分区：
文献类型：
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作者：
Y. Myer;Swatantar Kumar
用停流技术研究了CT-细胞色素的抗坏血酸还原作用。CT-细胞色素是两种化学生成的马心细胞色素C，FII和FII，含有蛋氨酸80和65，作为蛋氨酸亚硫醚，没有铁-硫键，以及不同于天然蛋白和彼此的电位和生理氧化还原特性(J.Pande等人，1987)。反应在550 nm处进行，研究在10 mM磷酸盐+0.17M氯化钠缓冲液中进行，pH为7.4。这两种CT-细胞色素都通过三相曲线减少，较快的和中间的抗坏血酸依赖的反应和缓慢的抗坏血酸非依赖的过程。两种CT-细胞色素都含有三种缓慢平衡的分子形式，两种通过与抗坏血酸反应直接还原，第三种通过转化为一种可还原形式。像天然蛋白质的反应一样，快速和中间过程对抗坏血酸的依赖都是非线性的，在高浓度时接近饱和值。假一级还原常数的抗坏血酸曲线是未修饰蛋白质还原反应的典型模型，结合后是一级还原反应(Myer等人，1980；Myer和Kumar，1984)，但具有不同的动力学参数、一级还原常数和蛋白质-抗坏血酸稳定常数。结果表明，在与抗坏血酸的还原反应中，两种CT-细胞色素之间的功能构象差异不具有任何显著的可操作性。蛋白质的蛋氨酸-80-硫铁连接不是蛋白质抗坏血酸还原的关键要求。该反应的主要机制也不敏感的蛋氨酸-80-S从血红素配位和/或相关的构象氧化还原性质的蛋白质。在反应的两个方面，即电子转移反应的效率和抗坏血酸阴离子-蛋白质复合体的稳定性中，前者依赖于分子结构构象状态的完整性。
The ascorbate reduction of the CT-cytochromes—two chemically generated forms of horse heart cytochrome c, FIII and FII, with both methionines, 80 and 65, as methionine sulfoxides, no iron-sulfur linkage, and potentiometric and physiological oxidoreduction properties distinct from those of the native protein and one another (J. Pande et al., 1987)—has been investigated using a stopped-flow technique. The reaction was monitored at 550 nm, and studies were conducted in 10 mM phosphate +0.17 M NaCl buffer,pH 7.4. Both CT-cytochromes are reduced by triphasic profiles, a faster and an intermediate ascorbate-dependent reaction and a slow, ascorbate-independent process. Both CT-cytochromes contain three molecular forms in slow equilibrium, two reducing directly by reaction with ascorbate and a third through conversion to one of the reducible forms. Like the reaction of the native protein, the ascorbate dependence of both the rapid and the intermediate process is nonlinear, approaching saturation values at high concentrations. The ascorbate profiles of the pseudo-first-order reduction constants are typical of the model for the reduction reaction of the unmodified protein, binding followed by a first-order reduction reaction (Myer et al., 1980; Myer and Kumar, 1984), but with distinct kinetic parameters, the first-order reduction constants and the protein-ascorbate stability constants. It has been concluded that the functional-conformational differences between the two CT-cytochromes are not operational to any significant extent in the reduction reaction with ascorbate. The methionine-80-sulfur-iron linkage of the protein is not a crucial requirement for the ascorbate reduction of the protein. The mechanism of the reaction in the main is also insensitive to the replacement of Met-80-S from heme coordination and/or the associated conformational-oxidoreduction properties of the protein. Of the two aspects of the reaction, the efficiency of the electron-transfer reaction and the stability of the ascorbate dianion-protein complex, the former is dependent on the integrity of the structural-conformational state of the molecule.