Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.
Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain.
复制标题
在细胞色素 bc1 复合物铁硫蛋白的系链区域用亮氨酸取代丙氨酸 86 会影响 [2Fe2S] 结构域的迁移率。
DOI：
10.1021/bi001708t
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Beattie,DS
中科院分区：
文献类型：
--
作者：
Ghosh,M;Wang,Y;Ebert,CE;Vadlamuri,S;Beattie,DS
突变酵母细胞色素bc 1复合物铁硫蛋白系链区中的三个保守丙氨酸残基导致酶活性降低22 - 56%[Obungu et al.（2000）Biochim. Biophys. Acta 1457，36 - 44]。与野生型相比，从A86 L分离的cytochromebc 1复合物的活性降低了60%，而没有血红素或蛋白质的损失，并且2Fe 2S簇或质子泵送能力没有变化。突变体A92 R的bc 1复合物的活性与野生型相同，而从突变体A90 I分离的bc 1复合物中观察到血红素和活性的损失。计算机模拟表明，突变A86 L和突变A92 R都不影响系链区的α-螺旋骨架;然而，取代Ala-86的亮氨酸侧链与Leu-89侧链相互作用。突变体A86 L的阿伦尼乌斯图显然是双相的，在17 - 19 °C下观察到转变，17 °C以下的活化能为279.9 kJ/mol，17 °C以上的活化能为125.1 kJ/mol。在突变体A86 L中，细胞色素1还原的初始速率降低了33%;然而，细胞色素b还原的初始速率不受影响，这表明铁硫蛋白的系链区的移动对于酶活性的最大速率是必要的。用亮氨酸取代Ala-86阻碍了α-螺旋的解旋，从而阻碍了系链的运动。
Mutating three conserved alanine residues in the tether region of the iron−sulfur protein of the yeast cytochromebc1complex resulted in 22−56% decreases in enzymatic activity [Obungu et al. (2000)Biochim. Biophys. Acta 1457, 36−44]. The activity of the cytochromebc1complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of thebc1complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in thebc1complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the α-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17−19 °C and an activation energy of 279.9 kJ/mol below 17 °C and 125.1 kJ/mol above 17 °C. The initial rate of cytochromec1reduction was lowered 33% in mutant A86L; however, the initial rate of cytochromebreduction was unaffected, suggesting that movement of the tether region of the iron−sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the α-helix and hence movement of the tether.