Expression and Analysis of Cyanobacterial Genes for the Cytochrome B6-f Complex

最近从蓝细菌Nostoc PCC 7906分离的基因 将与分子遗传学和生物化学一起使用， 蓝藻的操作，以调查方面的 细胞色素b6-f复合物的结构和功能。 这 膜蛋白复合物（Membrane protein complex） 质体喹啉-质体蓝素/细胞色素C氧化还原酶）是 光合作用电子传递和能量 叶绿体和蓝细菌中的转导。 未解决 问题包括电子转移和质子的途径 通过这些复合物的转运， 辅基，结合位点的性质， 醌底物，和功能的分子基础 b6-f和相关的材料类型之间的差异 b-Cl细胞色素复合物。 基因分析的障碍 细胞色素复合物是其在蓝藻形成中重要作用 光合和呼吸的电子传递。 因此一个 研究的目标是建立有条件的 携带潜在的衰弱突变的基因的表达。 具体目标是：1）表达念珠藻基因， Rieske Fe-S蛋白在大肠杆菌和大肠杆菌中的复合物 体外重构来自所述催化剂的催化活性络合物， 克隆的蛋白质和里斯克耗尽的复合物。 2)诱变 Rieske蛋白的Fe-S结合结构域，随后是 体外复溶以确定可能的S和N的性质 Fe-S中心的配体。 3)转录的确定 两个念珠藻细胞色素复合物操纵子的起始位点， 突变基因在厌氧条件下可能的条件表达 固氮条件。 4)念珠藻细胞色素的表达 蓝细菌Synechococcus PCC 7002中的复杂基因， 突变基因的可能条件分析。 5)随机 诱变所述亚基-IV蛋白的基因区域， 确定氨基酸残基的身份和作用 参与对苯二酚氧化。 蓝细菌和叶绿体有一个基本相似的 光合器 然而，蓝藻基因与 叶绿体的基因可以通过基因操作， 被改变的基因所取代 这些技术可以让基因 蛋白质结构/功能的解剖， 补充生物化学和生物物理方法。 拟议 研究将有助于阐明结构， 在细胞色素b6-f复合物中的功能， 基因分析尚未应用。 一个所述反应 这种复杂的，喹啉氧化，是限速步骤， 光合电子传递 因此基因操作 喹啉氧化位点的蛋白质结构可能导致 提高作物的光合效率，或 具体除草剂的应用。 该项目 此外，对一般理解 生物体内的电子传递和能量转换过程 膜。

Recently isolated genes from the cyanobacterium Nostoc PCC 7906 will be used together with molecular genetic and biochemical manipulations of cyanobacteria to investigate aspects of the structure and function of the cytochrome b6-f complex. This membrane protein complex (the plastoquinol-plastocyanin/cytochrome c oxidoreductase) is essential for photosynthetic electron transport and energy transduction in chloroplasts and cyanobacteria. Unresolved problems include the pathways of electron transfer and proton translocation through these complexes, the ligands for some of the prosthetic groups, the nature of binding sites for the quinone substrates, and the molecular basis of functional differences between the b6-f and the related mitochondrial-type b-cl cytochrome complexes. An obstacle to genetic analysis of the cytochrome complex is its essential role in cyanobacteria for both photosynthetic and respiratory electron transport. Thus one goal of the research is to establish means for the conditional expression of genes bearing potentially debilitating mutations. The specific goals are: 1) Expression of the Nostoc gene for the Rieske Fe-S protein of the complex in Escherichia coli and in vitro reconstitution of a catalytically active complex from the cloned protein and a Rieske-depleted complex. 2) Mutagenesis of the Fe-S binding domain of the Rieske protein followed by in vitro reconstitution to determine the nature of possible S and N ligands for the Fe-S center. 3) Determination of transcription start sites of the two Nostoc cytochrome complex operons for possible conditional expression of mutated genes under anaerobic N2-fixing conditions. 4) Expression of the Nostoc cytochrome complex genes in the cyanobacterium Synechococcus PCC 7002 for the possible conditional analysis of mutated genes. 5) Random mutagenesis of a region of the gene for the subunit-IV protein to determine the identities and roles of amino acid residues involved in quinol oxidation. Cyanobacteria and chloroplasts share a fundamentally similar photosynthetic apparatus. Cyanobacterial genes, however, unlike those of chloroplasts, can be manipulated genetically and replaced with altered genes. These techniques allow the genetic dissection of protein structure/function and powerfully complement biochemical and biophysical approaches. The proposed research would contribute to the elucidation of structure and function in the cytochrome b6-f complex to which molecular genetic analyses have not yet been applied. One of the reactions of this complex, quinol-oxidation, is the rate limiting step in photosynthetic electron transport. Thus genetic manipulations of protein structure at the quinol-oxidation site might lead to improvements in the photosynthetic efficiency of crop plants or to applications for specific herbicides. The project in addition has significance to the general understanding of electron transfer and energy transduction processes in biological membranes.