ISOLATION OF ELECTRON TRANSPORT COMPONENTS FROM MICROCYSTIS AERUGINOSA AND OSCILLATORIA SP

Abstract

Cyanobacteria are the most primitive organisms that possess an oxygenic photosynthetic system and the study of the cyanobacterial photosynthetic electron transport chain provides a useful model for higher plant systems. In this project, cytochrome c 553, plastocyanin and ferredoxin, all water-soluble components of the cyanobacterial electron transport chain, were isolated and purified from Microcystis aeruginosa. Cytochrome c 553 was purified from Oscillatoria sp. for reasons of comparison. Finally, cytochrome c550, an enigmatic haem protein whose function is unclear, was also purified from M- aeruginosa. A newly developed chromatographic procedure, using high concentrations of ammonium sulphate to fractionate water-soluble proteins on Sephacryl, was successfully employed together with conventional chromatographic tools to purify the proteins of interest to homogeneity. Sephacryl is a gel matrix normally used for gel filtration, but proteins can be caused to adsorb to it under high salt conditions. The proteins could then be selectively eluted by decreasing the salt concentration. As the adsorption of the proteins is independent of their molecular weights and pI values, it is suggested that the adsorption is based on hydrophobic interactions. Purified cytochrome c553 from M- aeruginosa (pI = 5.3) was separated by adsorption chromatography into four peaks which were identical with respect to adsorption spectrum, charge and molecular weight. Similarly, Oscillatoria sp. Cytochrome c553 (pI-9.3) was reproducibly eluted from the high salt chromatographic column as two peaks. The different forms were not due to amino acid modifications caused by high ammonium sulphate treatment. Since the cytochromes were purified from isogenic cultures, these forms may represent isozymes. Purified ferredoxin was eluted from the high salt adsorption column as two well-separated peaks. Although the two forms were identical with regards to molecular weight, spectrum and charge, they exhibited different mobilities during non-denaturing gel electrophoresis. Closely related isozymes are suggested. Purified cytochrome c550 was eluted as a single peak after cytochrome c553 from high salt chromatography columns. Cytochrome c550 was less soluble than cytochrome c553 in ammonium sulphate solutions and less heat-resistant, although at 100 °c, the adsorption of cytochrome c550 at 550 nm was only reduced by 50%. The biophysical nature of this cytochrome is discussed. Plastocyanin and cytochrome c553 are functional equivalents in the electron transport chain but plastocyanin is only synthesized when copper is available. M. aeruginosa was grew well in the presence of copper under alkaline conditions (pH=8.6), while exhibiting copper-induced suppression of cytochrome c553 • An acidic plastocyanin was isolated and purified from large scale Microcystis cultures. When the crude cell extracts were chromatographed on high salt Sephacryl columns, plastocyanin was eluted much earlier than the main contaminant, phycobiliproteins. Plastocyanin was also well resolved from cytochrome c553, which possesses similar molecular weight and charge characteristics. It was therefore possible for the first time, to purify an acidic cynaobacterial plastocyanin to homogeneity. Comparisons were made with purified Brassica chinensis plastocyanin. While Brassica plastocyanin exhibited a typical absorption maxima at 597 nm, M. aeruginosa plastocyanin has an unusual maxima at 600 nm. Microcystis plastocyanin was also less acidic (pI=4. 8) than higher plant plastocyanin (pI=3. 2) and adsorbed more strongly to high salt Sephacryl columns.