Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase

Bifunctional catalase-peroxidases are the least understood type of peroxida ses. A high-level expression in Escherichia coli of a fully active recombin ant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported. Since both physical and kine tic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the sp ectral characteristics and the reactivity of its redox intermediates by usi ng the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K-m = 4.8 mM and appa rent k(cat) = 8850 s(-1)). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M-1 S-1 at pH 7.0 and 15 deg rees C and the dissociation constant of the cyanide complex is 17 mu M. Bec ause of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for form ation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/ - 0.3) x 10(4) M-1 s(-1) at pH 7.0 and 15 degrees C. The spectrum of compou nd I is characterized by about 40% hypochromicity, a Soret region at 406 nm , and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rare constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M-1 s(-1), (5.4 +/- 0.3) x 10(5) M-1 s(-1), (1.6 +/- 0.3) x 10(5) M-1 s(-1) an d (4.3 +/- 0.2) x 10(4) M-1 s(-1), respectively. The redox intermediate for med upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferry l oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not e lectronically coupled to the heme. (C) 2000 Societe francaise de biochimie et biologie moleculaire I Editions scientifiques et medicales Elsevier SAS.