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

最近从蓝藻Nostoc PCC 7906中分离的基因将与蓝藻的分子遗传学和生化操作一起用于研究细胞色素b6-f复合物的结构和功能方面。这种膜蛋白复合物（质体醌-质体青素/细胞色素c氧化还原酶）对叶绿体和蓝藻的光合电子传递和能量转导至关重要。尚未解决的问题包括通过这些复合物的电子转移和质子易位的途径，一些配体基的配体，醌底物结合位点的性质，以及b6-f和相关线粒体型b-cl细胞色素复合物之间功能差异的分子基础。细胞色素复合体的遗传分析的一个障碍是它在蓝藻中光合作用和呼吸电子传递的重要作用。因此，这项研究的一个目标是建立有条件表达基因的方法，这些基因具有潜在的使人衰弱的突变。具体目标是：1)在大肠杆菌中表达Rieske Fe-S蛋白的Nostoc基因，并从克隆蛋白和Rieske- s缺失复合物中体外重建具有催化活性的复合物。2)对Rieske蛋白的Fe-S结合域进行诱变，然后在体外重建以确定Fe-S中心可能的S和N配体的性质。3)测定两个Nostoc细胞色素复合体操纵子的转录起始位点，研究在厌氧n2固定条件下突变基因可能的条件表达。4)在聚藻球菌PCC 7002中表达Nostoc细胞色素复合物基因，为突变基因的条件分析提供可能的条件。5)对亚单位- iv蛋白的基因区域进行随机诱变，以确定参与喹啉氧化的氨基酸残基的身份和作用。蓝藻和叶绿体共享一个基本相似的光合装置。然而，与叶绿体的基因不同，蓝藻的基因可以被基因操纵，并用改变的基因代替。这些技术允许对蛋白质结构/功能进行遗传解剖，并有力地补充了生物化学和生物物理方法。本研究将有助于阐明细胞色素b6-f复合物的结构和功能，而分子遗传学分析尚未对其进行应用。这种复合物的一个反应，喹啉氧化，是光合作用电子传递的限速步骤。因此，对喹啉氧化位点的蛋白质结构进行遗传操作可能会提高作物的光合效率或应用于特定的除草剂。此外，该项目对了解生物膜中的电子传递和能量转导过程具有重要意义。

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.