Functional and structural analysis of catalase oxidized by singlet oxygen.
Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.
Díaz
Adelaida
A
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D.F., Mexico.
Muñoz-Clares
Rosario A
RA
Rangel
Pablo
P
Valdés
Victor-Julián
VJ
Hansberg
Wilhelm
W
D002374:Catalase / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D026082:Singlet Oxygen / Q000737:chemistry*; D013172:Spores, Fungal / Q000201:enzymology*
D026082:Singlet Oxygen; D006418:Heme; D002374:Catalase