The Reaction of Synechocystis Catalase–Peroxidase (KatG) with Isoniazid Investigated by Multifrequency (9–285 GHz) EPR Spectroscopy

Abstract

Three distinct electron paramagnetic resonance (EPR) spectra of radical intermediates formed as reactive intermediates in the catalytic cycle of Synechocystis catalase–peroxidase were identified. Multifrequency EPR spectroscopy, combined with site-directed mutagenesis and selective deuterium labeling of Trp and Tyr residues, allowed us to unequivocally assign such intermediates to an [Fe(IV) = O Por^·+] species, the first committed intermediate in monofunctional peroxidases and two protein-based radicals, identified as Trp106^· and a Tyr^·, formed subsequently to the [Fe(IV) = O Por^·+] species by intramolecular electron transfer. Our recent characterization of the Mycobacterium tuberculosis catalase–peroxidase showed that the Trp^· sites differ among these enzymes, and that the [Fe(IV) = O Trp^·] species was the reactive intermediate with the prodrug isoniazid. Accordingly, the question to address was whether the dissimilarity in the sites for the formation of the Trp^· intermediates and in the geometry of the distal side was reflected by differences in the peroxidase-like reaction of Synechocystis and Mycobacterium tuberculosis catalase–peroxidases with the prodrug isoniazid. Our findings show that in the Synechocystis enzyme, the isoniazid substrate can get closer to the heme distal side and can react readily with the [Fe(IV) = O Por^·+] species, at variance to the situation in the M. tuberculosis catalase–peroxidase. These results indicate that, as in the case of monofunctional peroxidases, the difference in the sites for the formation of the Trp^· as alternative reactive intermediates to the [Fe(IV) = O Por^·+] species is correlated to differences in substrate binding sites.