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 f orm of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis P CC 6803 is reported. Since both physical and kinetic characterization revea led its identity with the wild-type protein, the large quantities of recomb inant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phe nols and anilines) by a bifunctional catalase-peroxidase compound I using t he sequential-mixing stopped-flow technique. Because of the overwhelming ca talase activity,peroxoacetic acid has been used for compound I formation. A greater than or equal to 50-fold excess of peroxoacetic acid is required t o obtain a spectrum of relatively pure and stable compound I which is chara cterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbe stic 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 ferri c 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 t he substituent effect on the benzene ring. The apparent second-order rate c onstants varied from (3.6 +/- 0.1) x 10(6) M-1 s(-1) for p-hydroxyaniline t o (5.0 +/- 0.1) x 10(2) M-1 s(-1) for p-hydroxybenzenesulfonic acid. They a re shown to correlate with the substituent constants in the Hammett equatio n, which suggests that in bifunctional catalase-peroxidases the aromatic do nor molecule donates an electron to compound I and loses a proton simultane ously. 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 m aximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent i n KatG compound II is contained on an amino acid which is not electronicall y coupled to the heme.