Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120.
The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.
Regelsberger
Günther
G
Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Atzenhofer
Werner
W
Ruker
Florian
F
Peschek
Gunter A
GA
Jakopitsch
Christa
C
Paumann
Martina
M
Furtmüller
Paul Georg
PG
Obinger
Christian
C
D000595:Amino Acid Sequence; D017033:Anabaena / Q000201:enzymology*; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D019897:Periplasm / Q000378:metabolism; D010802:Phylogeny; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*; D020524:Thylakoids / Q000378:metabolism
D008345:Manganese; D013482:Superoxide Dismutase