Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata.
Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.
D001386:Azides / Q000494:pharmacology; D002374:Catalase / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000032:analysis; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D011190:Potassium Cyanide / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D012241:Rhodopseudomonas / Q000201:enzymology*; D019810:Sodium Azide; D013053:Spectrophotometry; D013816:Thermodynamics
D001386:Azides; D006898:Hydroxylamines; D046911:Macromolecular Substances; D019811:Hydroxylamine; D006418:Heme; D019810:Sodium Azide; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002374:Catalase; D011190:Potassium Cyanide