title: Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings.
abstract: 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 second 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 Tyr(229). 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 Tyr(229) 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 Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.
authors.affiliation: Department of Chemistry, Brooklyn College, Brooklyn, New York 11210, USA.
authors.forename: Kalina
authors.initials: K
authors.lastname: Ranguelova
authors.forename: Stefania
authors.initials: S
authors.lastname: Girotto
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Gerfen
authors.forename: Shengwei
authors.initials: S
authors.lastname: Yu
authors.forename: Javier
authors.initials: J
authors.lastname: Suarez
authors.forename: Leonid
authors.initials: L
authors.lastname: Metlitsky
authors.forename: Richard S
authors.initials: RS
authors.lastname: Magliozzo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002374:Catalase / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D004579:Electron Transport; D005609:Free Radicals / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005609:Free Radicals; D014443:Tyrosine; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis