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

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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.