Purification and characterization of a hydroperoxidase from the cyanobacterium Synechocystis PCC 6803: identification of its gene by peptide mass mapping using matrix assisted laser desorption ionization time-of-flight mass spectrometry

A cytosolic catalase-peroxidase from the cyanobacterium Synechocystis PCC 6 803 was purified to homogeneity by a six-step purification procedure. It is a homodimeric enzyme with a subunit molecular mass of 85 kDa. The isoelect ric point of the protein is at pH 5.5; Michaelis constant, turnover number, and catalytic efficiency of the catalase activity for H2O2 were measured t o be 4.8 mM, 3450 s(-1), and 7.2x10(5) M-1 s(-1), respectively. Preparation and spectroscopy of the pyridine ferrohemochrome identified an iron protop orphyrin IX as the prosthetic group. The enzyme was shown to exhibit both c atalase and peroxidase activities, both of which were inhibited by cyanide, leading to a high-spin to low-spin transition of the heme iron center as d etected by a shift of the Soret peak from 405 to 421 nm. The catalase-speci fic inhibitor 3-amino-1,2,4-triazole proved ineffective. o-Dianisidine, pyr ogallol and guaiacol functioned as a peroxidatic substrate, but no reaction was detected with NADH, NADPH, glutathione, and ascorbate. Peptide mass ma pping using matrix assisted laser desorption ionization time-of-flight mass spectrometry showed the identity between the purified protein and a putati ve katG gene derived from the genome of SI Synechocystis PCC 6803. A compar ison of amino acid sequences of the catalase-peroxidase from Synechocystis PCC 6803 and those from other bacteria showed a high homology around the as sumed distal and proximal histidine residues, suggesting a highly conserved histidine as the fifth ligand of the heme iron. (C) 1999 Federation of Eur opean Microbiological Societies. Published by Elsevier Science B.V. All rig hts reserved.