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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.

SUBMITTER: Ranguelova K

PROVIDER: S-EPMC1885898 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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

Ranguelova Kalina K Girotto Stefania S Gerfen Gary J GJ Yu Shengwei S Suarez Javier J Metlitsky Leonid L Magliozzo Richard S RS

The Journal of biological chemistry 20070104 9

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 electr ...[more]

PMID: 17204474

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Project description:The validity of the popular point-dipole approximation for interpretation of the zero-field splitting (ZFS) parameter (D-value) in EPR spectroscopy is studied. This approximation is of central importance for the determination of distances by analysis of EPR data. In this work, a detailed experimental (EPR spectroscopy and X-ray crystallography) and theoretical study for a model system (2,2',5,5'-tetra(tert-butyl)-4,4'-bis(ethoxy-carbonyl)-3,3'-bipyrrolyl-1,1'-dioxyl) was performed to understand the scope and limitations of the point-dipole model in EPR spectroscopy. For this diradical, the radical-radical distance derived with the point-dipole approximation deviates significantly (by approximately 40%) from the results derived from the X-ray analysis. Explicit quantum chemical calculation of the D-value on the basis of B3LYP density functional calculations leads to excellent quantitative agreement with the measured D-value. The quantitative accuracy of the employed methodology was confirmed for two additional systems that have previously been experimentally characterized. We therefore analyzed the contributions to the D-value of the target system in detail. This analysis leads to insight into the reasons for the failure of the point-dipole approximation. The analysis was then extended to an in silico study of five classes of model systems. Linkers of varying length and bond saturation were introduced between the radical-carrying groups. This allows for the analysis of the distance dependence of the D-parameter as well as the through-bond and through-space spin-spin interaction. From these results we established the limitations of the point-dipole approximation. The results of this analysis demonstrate that even very modest amounts of spin delocalization can cause significant deviations from pure point-dipole behavior and consequently cause the EPR derived distances to deviate from the N-O midpoint distance by up to several angstroms. If unsaturated linkers are used, the distance dependence of D does not follow the inverse cubic behavior predicted by the point-dipole model. However, for commonly used nonaromatic nitroxide rings connected by a saturated linker, the point-dipole approximation works well. Among the various point-dipole variants tested in this work for delocalized spins, the most successful one is based on distributed point-dipoles with spin populations derived from quantum chemical calculations. The distance dependence of the isotropic Heisenberg exchange parameter J has also been studied theoretically. The decay was found to be monoexponential with a decay constant of approximately 1 A(-1). Thus at linker lengths between 6-8 carbon atoms between a nitroxide radical pair, a switch from the strong to the weak exchange limit is predicted.

Dataset Information

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

Publications

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

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