Tian H, Yu L, Mather M W, Yu C A
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
J Biol Chem. 1998 Oct 23;273(43):27953-9. doi: 10.1074/jbc.273.43.27953.
The crystal structure of the mitochondrial cytochrome bc1 complex suggests that movement of the extramembrane (head) domain of the Rieske iron-sulfur protein (ISP) is involved in electron transfer. Such movement requires flexibility in the neck region of ISP. To test this hypothesis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with altered ISP necks (residues 39-48) were generated and characterized. Mutants with increased rigidity of the neck, generated by a double-proline substitution at Ala-46 and Ala-48 (ALA-PLP) or by a triple-proline substitution of ADV at residues 42-44 (ADV-PPP), have retarded (50%) or no photosynthetic growth, respectively. However, the mutant with a shortened neck, generated by deleting ADV (DeltaADV), has a photosynthetic growth rate comparable to that of complement cells, indicating that the length of the ISP neck is less critical than its flexibility in support of photosynthetic growth. The DeltaADV and ALA-PLP mutant membranes have 10 and 30% of the cytochrome bc1 complex activity found in the complement membrane, respectively, whereas the ADV-PPP mutant membrane contains no cytochrome bc1 complex activity. The loss of cytochrome bc1 complex activity in the DeltaADV membrane is attributed to improper docking of the head domain of ISP on cytochrome b, as indicated by a drastic change in the EPR characteristics of the Rieske iron-sulfur cluster. The loss of cytochrome bc1 complex activity in the ALA-PLP and ADV-PPP mutant membranes results from the decreased mobility of the ISP head domain due to the increased rigidity of the ISP neck. The ALA-PLP mutant complex has a larger activation energy than the wild-type complex, suggesting that movement of the head domain decreases the activation energy barrier of the cytochrome bc1 complex. Using the conditions developed for the isolation of the His-tagged complement cytochrome bc1 complex, a two-subunit complex (cytochromes b and c1) was obtained from the DeltaADV and ADV-PPP mutants, indicating that mutations at the neck region of ISP weaken the interactions among cytochrome b, ISP, and subunit IV.
线粒体细胞色素bc1复合物的晶体结构表明， Rieske铁硫蛋白（ISP）的膜外（头部）结构域的移动参与了电子传递。这种移动需要ISP颈部区域具有灵活性。为了验证这一假设，我们构建并表征了球形红杆菌突变体，这些突变体表达带有His标签的细胞色素bc1复合物，其ISP颈部（第39 - 48位氨基酸残基）发生了改变。通过在Ala - 46和Ala - 48处进行双脯氨酸取代（ALA - PLP）或在第42 - 44位氨基酸残基处对ADV进行三脯氨酸取代（ADV - PPP）而产生的颈部刚性增加的突变体，分别具有光合生长延迟（50%）或无光合生长的现象。然而，通过缺失ADV（DeltaADV）产生的颈部缩短的突变体，其光合生长速率与互补细胞相当，这表明ISP颈部的长度在支持光合生长方面不如其灵活性关键。DeltaADV和ALA - PLP突变体膜中细胞色素bc1复合物的活性分别为互补膜中的10%和30%，而ADV - PPP突变体膜中则不含有细胞色素bc1复合物活性。DeltaADV膜中细胞色素bc1复合物活性的丧失归因于ISP头部结构域与细胞色素b的对接不当，这由Rieske铁硫簇的EPR特征的剧烈变化所表明。ALA - PLP和ADV - PPP突变体膜中细胞色素bc1复合物活性的丧失是由于ISP颈部刚性增加导致ISP头部结构域的移动性降低。ALA - PLP突变体复合物比野生型复合物具有更大的活化能，这表明头部结构域的移动降低了细胞色素bc1复合物的活化能垒。利用为分离带有His标签的互补细胞色素bc1复合物所开发的条件，从DeltaADV和ADV - PPP突变体中获得了一个二聚体复合物（细胞色素b和c1），这表明ISP颈部区域的突变削弱了细胞色素b、ISP和亚基IV之间的相互作用。
