Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Kalina Ranguelova, Stefania Girotto, Gary J. Gerfen, Shengwei Yu, Javier Suarez, Leonid Metlitsky, Richard S. Magliozzo

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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 Tyr229. 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 Tyr229 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 Tyr353, which lies more distant from the heme. The 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.

Original languageEnglish (US)
Pages (from-to)6255-6264
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number9
DOIs
StatePublished - Mar 2 2007

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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