Purification and characterization of a homodimeric catalase-peroxidase from the cyanobacterium Anacystis nidulans.
Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Km for H2O2 of 4.3 mM, a calculated turnover number of 7200 s(-1), and an overall-rate constant of 3.5 x 10(6) M(-1) s(-1). The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki = 27.2 microM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2 than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.
Obinger
C
C
Institut für Chemie, Universität für Bodenkultur, Wien, Austria. cobinger@edv2.boku.ac.at
Regelsberger
G
G
Strasser
G
G
Burner
U
U
Peschek
G A
GA
D000640:Amitrole / Q000494:pharmacology; D001426:Bacterial Proteins; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003486:Cyanides / Q000494:pharmacology; D000458:Cyanobacteria / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D004227:Dithionite; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D010544:Peroxidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003486:Cyanides; D046911:Macromolecular Substances; D004227:Dithionite; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D000640:Amitrole