Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex.
Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex.
复制标题
细胞色素 bc1 复合物中工程亚基间二硫键的形成会破坏复合物中的电子转移活性。
DOI：
10.1016/j.bbabio.2008.01.005
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Yu,Chang-An
中科院分区：
文献类型：
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作者：
Ma,He-Wen;Yang,Shaoqing;Yu,Linda;Yu,Chang-An
Rieske铁硫蛋白的蛋白质结构域运动被推测在由细胞色素bc 1复合物催化的泛醇的分叉氧化中发挥重要作用。为了更好地理解在Qp位点的分叉泛醇氧化的电子转移机制，我们通过在两个基因工程半胱氨酸残基之间创建亚基间二硫键将ISP的头部结构域固定在cyt c1位置：一个在ISP的位置141，另一个在cyt c1的位置180 [S141 C（ISP）/G180 C（cyt c1）]。SDS-PAGE和Western印迹证实该突变复合物中ISP和cyt c1之间形成了二硫键。在该突变体复合物中，二硫键的形成与复合物的电子转移活性的丧失同时发生。当用β-巯基乙醇处理释放二硫键时，活性恢复。这些结果进一步支持了ISP头部结构域的流动性在细胞色素bc 1复合物中具有重要功能的假设。ISP与cyt c1之间形成二硫键，缩短了[2Fe-2S]簇与血红素c1之间的距离，从而提高了pH变化引起的两个氧化还原辅基之间的电子转移速率。亚基间二硫键的形成也降低了率的豆磷脂诱导还原ISP在完全氧化的复合物，这表明内源性电子供体来自附近的B的位置在细胞色素B。
Protein domain movement of the Rieske iron–sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1[S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1complex. Formation of the disulfide bond between ISP and cyt c1shortens the distance between the [2Fe–2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.