RADICAL SITES IN M. TUBERCULOSIS KATG IDENTIFIED USING EPR SPECTROSCOPY, THE 3-D CRYSTAL STRUCTURE AND ELECTRON-TRANSFER COUPLINGS

• Published in: The Journal of biological chemistry 2007-01, Vol.282 (9), p.6255-6264
• Main Authors: Ranguelova, Kalina, Girotto, Stefania, Gerfen, Gary J., Yu, Shengwei, Suarez, Javier, Metlitsky, Leonid, Magliozzo, Richard S.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M607309200

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis , a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates, and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid based radicals during turnover with alkyl peroxides and this work focuses on extending the identification and characterization of radicals forming on the millisecond to seconds time scale. Rapid freeze- quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme-pocket binding of the drug, and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Y229. High-field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal and new analyses of X-band RFQ-EPR spectra also established its presence. High-field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on X-ray structural data for particular tyrosine and tryptophan residues enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Y229 within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species, and finally to only a tyrosyl radical on residue Y353, which lies more distant from the heme. Radical processing of enzyme lacking the Trp107-Tyr229-Met255 adduct, found as a unique structural feature of catalase-peroxidases, is suggested to be a reasonable assignment of the phenomena.