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) × 103 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) × 106 M-1 s-1 for p-hydroxyaniline to (5.0 ± 0.1) × 102 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 ρ, 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.