Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I.
A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm. The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7. 0. Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring. The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid. They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously. The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines. The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.
Regelsberger
G
G
Institute of Chemistry, University of Agricultural Sciences, Vienna, Austria.
Jakopitsch
C
C
Engleder
M
M
Rüker
F
F
Peschek
G A
GA
Obinger
C
C
D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D013053:Spectrophotometry; D013057:Spectrum Analysis
D000814:Aniline Compounds; D001426:Bacterial Proteins; D010636:Phenols; D011994:Recombinant Proteins; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI