TY - JOUR
T1 - Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings
AU - Ranguelova, Kalina
AU - Girotto, Stefania
AU - Gerfen, Gary J.
AU - Yu, Shengwei
AU - Suarez, Javier
AU - Metlitsky, Leonid
AU - Magliozzo, Richard S.
PY - 2007/3/2
Y1 - 2007/3/2
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M607309200
DO - 10.1074/jbc.M607309200
M3 - Article
C2 - 17204474
AN - SCOPUS:34250328351
SN - 0021-9258
VL - 282
SP - 6255
EP - 6264
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 9
ER -