Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants.
Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H(2)O(2), is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 degrees C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2x10(4)M(-1)s(-1), 30M(-1)s(-1), 3.4x10(3)M(-1)s(-1), and 8.6x10(3)M(-1)s(-1), respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.
Vlasits
Jutta
J
Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria.
Furtmüller
Paul G
PG
Jakopitsch
Christa
C
Zamocky
Marcel
M
Obinger
Christian
C
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003486:Cyanides; D050505:Mutant Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria