Respiratory electron transport in cyanobacteria

Cyanobacteria are the largest, most diversified, evolutionary most significant, and ecologically most successful micro-organisms on our earth. They were the first and are up to now the only oxygenic phototrophic prokaryotes having uniquely accomodated both oxygenic photosynthesis and aerobic respiration within a single prokaryotic cell. The thylakoid membrane (ICM) shares a number of components of both redox pathways such as the plastoquinone pool and cytochrome b6 f, while the cytoplasmic membrane (CM) only contains a respiratory electron transport chain but no photosynthetic reaction centers. However, in contrast to eukaryotic organisms, the knowledge of cyanobacterial respiratory electron transport chains is insufficient in order to understand its role in an oxygenic phototrophic bacterium. Analysis of entire genomes of cyanobacteria showed the existence of genes encoding an aa 3 -type cytochrome c oxidase (CcO). A respiratory cytochrome c is lacking in cyanobacteria and it has been proposed that these organisms use cytochrome c 6 and plastocyanin (a type I copper protein) to serve both in photosynthesis and respiration. In all cyanobacteria studied so far cytochrome c 6 and plastocyanin are functionally interchangeable and share a number of similar physicochemical and structural features that allow them to interact with the same redox partners. In addition a new c-class cytochrome, the so-called cytochrome c M, has been found in cyanobacteria, but its physiological function is still unknown. It is the aim of this work to investigate the role of cytochrome c 6 , cytochrome c M and plastocyanin in cyanobacterial respiration. In detail, the interprotein electron transfer between these putative donors and membranous CcO from isolated CM and ICM as well as with the recombinant truncated subunit II copper A domain of CcO, which is known to be the electron entry and donor binding site, will be investigated. By using site- directed mutagenesis in combination with multimixing stopped-flow kinetic techniques and NMR spectroscopy the interaction modes between the putative electron-donating metalloproteins and CcO from Synechocystis PCC 6803, Anabaena PCC 7119 and Phormidium laminosum will be elucidated. Since both charged and hydrophobic areas could contribute to the protein interaction, the dependence of rates on ionic strength and pH has to be investigated, since topographical models suggest that the copper A domain can be located in two completely different environments, namely the periplasmic and thylakoid luminal spaces.

Cyanobacteria are the largest, most diversified, evolutionary most significant, and ecologically most successful micro-organisms on our earth. They were the first and are up to now the only oxygenic phototrophic prokaryotes having uniquely accomodated both oxygenic photosynthesis and aerobic respiration within a single prokaryotic cell. The thylakoid membrane (ICM) shares a number of components of both redox pathways such as the plastoquinone pool and cytochrome b6 f, while the cytoplasmic membrane (CM) only contains a respiratory electron transport chain but no photosynthetic reaction centers. However, in contrast to eukaryotic organisms, the knowledge of cyanobacterial respiratory electron transport chains is insufficient in order to understand its role in an oxygenic phototrophic bacterium. Analysis of entire genomes of cyanobacteria showed the existence of genes encoding an aa 3 -type cytochrome c oxidase (CcO). A respiratory cytochrome c is lacking in cyanobacteria and it has been proposed that these organisms use cytochrome c 6 and plastocyanin (a type I copper protein) to serve both in photosynthesis and respiration. In all cyanobacteria studied so far cytochrome c 6 and plastocyanin are functionally interchangeable and share a number of similar physicochemical and structural features that allow them to interact with the same redox partners. In addition a new c-class cytochrome, the so-called cytochrome c M, has been found in cyanobacteria, but its physiological function is still unknown. It is the aim of this work to investigate the role of cytochrome c 6 , cytochrome c M and plastocyanin in cyanobacterial respiration. In detail, the interprotein electron transfer between these putative donors and membranous CcO from isolated CM and ICM as well as with the recombinant truncated subunit II copper A domain of CcO, which is known to be the electron entry and donor binding site, will be investigated. By using site- directed mutagenesis in combination with multimixing stopped-flow kinetic techniques and NMR spectroscopy the interaction modes between the putative electron-donating metalloproteins and CcO from Synechocystis PCC 6803, Anabaena PCC 7119 and Phormidium laminosum will be elucidated. Since both charged and hydrophobic areas could contribute to the protein interaction, the dependence of rates on ionic strength and pH has to be investigated, since topographical models suggest that the copper A domain can be located in two completely different environments, namely the periplasmic and thylakoid luminal spaces.