Abstract
The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical approximately 0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for l-3,3-[2H2]tyrosine-labeled, but not l-2,4,5,6,7-[2H5]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.
Highlights
Catalase-peroxidases are multimeric heme enzymes found in bacteria and other microorganisms
Rapid freeze-quench EPR spectroscopy experiments combined with characterization of the observed EPR spectra and isotopic substitution experiments demonstrated the formation of tyrosyl radicals in M. tuberculosis KatG in reactions with peroxyacetic acid
Identification of Tyrosyl Radical—Fig. 1 shows the progress of the reaction of ferric M. tuberculosis KatG with peroxyacetic acid from 50 ms to 10 s after mixing in a typical rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiment (g ϭ 2 region)
Summary
KatG, catalase-peroxidase; INH, isoniazid (isonicotinic acid hydrazide); Cmpd I, compound I; Cmpd II, compound II; PAA, peroxyacetic acid; PGHS, prostaglandin H synthase; RFQ-EPR, rapid-freeze-quench electron paramagnetic resonance. KatG Cmpd I decays relatively rapidly in the absence of exogenous substrates, suggesting that endogenous electron transfers produce amino acid-based radicals during the process This idea had been suggested from optical experiments on other catalase-peroxidases [15,16,17,18], in which an intermediate labeled Compound II, but exhibiting an optical spectrum closely resembling the resting enzyme, was suggested to contain an unidentified oxidized amino acid that is not electronically coupled to the heme. In an effort to investigate this possibility and further characterize the catalytic pathway in KatG, we have undertaken rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) spectroscopy experiments In this approach, the resting (ferric) enzyme is mixed with peroxide and allowed to react for various periods of time, followed by rapid freezing of the mixture to quench the reaction; EPR spectroscopy is used to examine the products. The observations presented here distinguish the behavior of this enzyme from other Class I peroxidases
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