PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC CATALASE-PEROXIDASE FROM THE CYANOBACTERIUM ANACYSTIS-NIDULANS

Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit bo th catalase and o-dianisidine peroxidase activity, Native polyacrylami de gel electrophoresis demonstrates one distinct enzyme, which has bee n purified to essential homogeneity and found to be composed of two id entical 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 betwe en 6.5 and 7.5 and a K-m for H2O2 of 4.3 mM, a calculated turnover num ber 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 (K-i = 27.2 mu M) and insensitive to the eukaryotic catalase inhibitor 3-ami no-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, bu t not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indi cating that Compound I is much faster reduced by H2O2 than it is forme d. The native enzyme is in the high-spin state, which is transformed t o 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 isosbe stic points at 354 and 427 nm. (C) 1997 Academic Press.