The Role of Supernumerary Subunit in the Bacterial BC1 Complex

0077650余本研究的总体目标是阐明的作用，在细胞色素bc 1复合物的光合细菌Rhodobacter sphaeroides的超数亚基，亚基IV（Mr = 14，384）。以前的研究涉及定点诱变，然后通过基因互补（体内重建）已经确定，亚基IV是必不可少的催化活性，Q-结合，和结构完整的复合物。然而，从细菌bc 1复合物的3-D结构模型来看，与线粒体对应物（细胞色素B、细胞色素c1、铁硫蛋白和亚基VII）的坐标一起构建，亚基IV直接参与复合物的催化（Q-结合）位点似乎不太可能。在本项目中，PI希望继续使用体外重构，结合生物物理、生物化学和结构分析，以探索亚基IV与核心bc 1复合物之间的相互作用模式。本项目的具体目标是：（a）鉴定与三亚基核心复合物相互作用的亚基IV的氨基酸残基;（B）鉴定参与与亚基IV相互作用的细胞色素B的氨基酸残基;（c）通过圆二色性和差示扫描量热法研究亚基IV对细胞色素bc 1复合物的蛋白质构象和热稳定性的影响;（d）研究蛋白质：三个和四个亚基复合物中的脂质相互作用;（e）使用合成的Q-衍生物检查亚基IV的Q螯合作用;（f）探索细菌核心亚基中的超数亚基功能与额外片段的可能关联。本项目的研究将增加我们对球形红细菌bc 1复合体的结构-功能和作用机制的理解。所获得的知识将提供一个更好地了解光合机构中的能量转导机制。 这可能会导致设计的方法，以提高光合效率和有效的除草剂设计。 它也将增强我们对线粒体生物能量学的理解。

0077650YuThe overall objective of this research is to elucidate the role of the supernumeral subunit, subunit IV (Mr = 14,384), in the cytochrome bc1 complex from a photosynthetic bacterium Rhodobacter sphaeroides. Previous studies involving site-directed mutagenesis followed by gene complementation (in vivo reconstitution) have established that subunit IV is essential for the catalytic activity, Q-binding, and structural integrity of the complex. However, judging from the 3-D structure model of this bacterial bc1 complex, built with coordinates from their mitochondrial counter parts (cytochrome b, cytochrome c1, the iron-sulfur protein, and subunit VII), direct participation of subunit IV in the catalytic (Q-binding) sites of the complex seems unlikely. In this project, the PI wishes to continue to use in vitro reconstitution, in concert with biophysical, biochemical, and structural analysis, to probe the mode of interaction between subunit IV and the core bc1 complex.The specific aims of this project are: (a) to identify amino acid residues of subunit IV involved in interaction with the three-subunit core complex; (b) to identify amino acid residues of cytochrome b involved in interaction with subunit IV; (c) to investigate the effect of subunit IV on the protein conformation and thermostability of the cytochrome bc1 complex by circular dichroism and differential scanning calorimetry; (d) to investigate the protein:lipid interactions in three- and four- subunit complexes; (e) to examine the Q sequestering role of subunit IV using synthetic Q- derivatives; and (f) to explore the possible association of supernumeral subunit function with extra segments in the bacterial core subunits. The research described in this project will increase our understanding of the structure-function and mechanisms of action of the Rhodobacter sphaeroides photosynthetic bacterial bc1 complex. The knowledge gained will provide a better understanding of energy transduction mechanisms in the photosynthetic apparatus. This may lead to the design of methods to improve photosynthetic efficiency and effective herbicide design. It will also enhance our understanding of mitochondrial bioenergetics.