Scavenging of Reactive Oxygen Spezies in Vegetative Cells and Protection of Nitrogenase in Heterocysts of the Cyanobacterium Anabaena variabilis ATCC 29413

Nitrogen-fixing cyanobacteria make a significant contribution to the global nitrogen cycle. Symbiotic and free- fiving marine cyanobacteria probably contribute more to global nitrogen fixation than do all the other nitrogen- fixing bacteria put together. The enzyme for nitrogen fixation is the nitrogenase. It is rapidly and irreversibly inactivated by molecular oxygen and reactive oxygen species. In this project Anabaena variabilis, a filamentous cyanobacterium that can fix nitrogen within specialized cells, the heterocysts, is selected to investigate the role of enzyme systems for scavenging of reactive oxygen species. In chloroplasts, toxic oxygen species are removed by ascorbate and an ascorbate-regenerating system, which includes superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase. It is tempting to speculate that a similar reaction cascade is used in cyanobacterial cells. A combination of chromatographic methods will be utilized to purify the enzymes. Biochemical analysis will follow to find out more about the respective enzymes: Localization, molecular mass and subunit composition, prosthetic groups; potential substrates, Michaelis constant; turnover number, catalytic efficiency, specific activity and overall-rate constant; influence of temperature, pH ionic strength,... on enzymatic activity, steady-state kinetics, inhibitors of enzymatic activity; rate constants of enzymatic intermediate formation; amino acid sequencing; preparation of polyclonal antibodies raised against (subunits of) purified enzyme and immunogold labeling. Besides it will be investigated which effects are caused at different growth conditions, such as high oxygen concentration, or addition of hydrogen peroxide or radical-forming agents. After incubation at various times, cells are broken and the extract will be tested for enzymatic activities. So the potential of adaptation to toxic oxygen species can be elucidated. Also the influence on nitrogenase activity will be examined under these conditions to find out which conditions are critical for viability of this enzyme.

Primordial blue-green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant-type photosynthetic organisms, thereby releasing huge amounts of molecular oxygen. Thus, it is reasonable to assume that cyanobacteria were among the first to adapt to the new, increasingly oxygen-rich environmemt and to elaborate different redox enzymes for scavenging of partially reduced toxic oxygen species (superoxide and hydrogen peroxide). We have screened several both unicellular and filamentous cyanobacteria for hydroperoxidases and superoxide dismutases. The enzymes have been isolated, the corresponding genes were cloned, overexpressed in E. coli and the spectral and kinetic features were analyzed. Generally, cyanobacteria contain two kinds of superoxide dismutase (SOD), namely iron- and/or manganese-SOD. The filamentous nitrogen fixing species Anabaena variabilis was chosen as a representative showing SOD activity in membrane preparations and cytosolic extracts. Western blotting and immunodetection applying antibodies depicts that the Fe-SOD is in the cytosol and the Mn-SOD membrane-bound. Their localization was also confirmed within vegetative cells and heterocysts by immunogold labeling and transmission electron microscopy. Both SODs were overexpressed in E. coli and purified to homogeneity. Cytosolic, dimeric Fe-SOD (2 23 kDa) has an isoelectric point at 5.9 and a specific activity of approximately 2000 units per mg. Stopped-flow measurements showed a clear pH dependence of the second-order rate constant; with increasing pH values the rate constant decreases. Whereas it is not inhibited by azide and cyanide, it is irreversibly inactivated by hydrogen peroxide. FeSOD was shown to be present is both vegetative cells and heterocysts at the same amount. The latter is the specialized cell type containing the oxygen-sensitive nitrogenase. Overexpression of the whole Mn-SOD was not successful. This may be due to the hydrophobic domain located at the N-terminus that is proposed to act as a membrane anchor. So, two soluble portions of the Mn-SOD were expressed, purified and characterized. The specific activity is rather low with about 500-600 units per mg. Rate constants decrease with increasing pH values and are lower than that of the Fe-SOD. Mn-SOD is localized in both the vegetative cells and heterocysts of the cyanobacterium. Crystallization of the catalytic domain was successful and the X-ray structure was solved at 2.0 Å resolution. Two types of heme hydroperoxidases are found in cyanobacteria. Bifunctional catalase-peroxidases (members of Class I of the plant peroxidase superfamily) and/or monofunctional catalases. Cyanobacteria do not contain ascorbate peroxidases. Interestingly, in Anabaena variabilis no heme-containing hydroperoxidase was found both on a genetic and biochemical approach. Another system for removing hydrogen peroxide was found, namely a thioredoxin peroxidases. Until now, two thioredoxin peroxidases from Synechocystis PCC 6803 and Anabaena variabilis have been characterized. The protein from Anabaena is an acidic, homodimeric cytosolic protein protein found in both vegetative cells and heterocysts. It is a peroxidase that reduces hydrogen peroxide with the use of thioredoxin as electron donor containing two redox-active cysteine residues. Kinetic and stability measurements were performed and crystallization of the recombinant protein is in progress.