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.

Fig. 1. Met-Tyr-Trp adduct in Mtb KatG

The figure was constructed using the coordinates deposited in the Protein Data Bank (accession code 2CCA) and displayed using PyMOL software.

Fig. 2. RFQ-EPR spectra of Mtb KatG

Resting enzyme (wild-type KatG, 50 μM final) was reacted with a mixture of INH (10 mM) and PAA (150 μM final) at 25 °C (narrow doublet). Incubation times before freezing are indicated. Inset: the experimental narrow and wide doublets with simulated spectra based on the parameters in Table 1.

Fig. 3. RFQ-EPR spectrum of KatG[W107F]

Resting enzyme (50 μM final) was reacted with PAA (150 μM final) at 25 °C and frozen after 6.4 ms reaction (top spectrum, narrow singlet). Also shown are spectra of wild type KatG frozen at the indicated reaction times after manual mixing (same concentrations): A – 10 sec (wide singlet), B – 2 min, C – 5 min (narrow singlet). The dotted spectrum is a simulation of the narrow singlet based on the parameters given in Table 1.

Fig. 4. High-field (130 GHz) EPR spectra of Mtb KatG

The 800 ms sample (bottom spectrum) was prepared using the RFQ-EPR apparatus modified to load the sample into a quartz capillary. Final concentrations: KatG, 300 μM, PAA, 900 μM. The 10-sec reaction sample (top spectrum) was mixed and loaded into the capillary manually. The g-tensor components of the tyrosyl radical and the tryptophanyl radical are indicated. The spectra are derivatives of echo-detected spectra obtained at 7 K. Other acquisition parameters are given in the Experimental Procedures.

Fig. 5. RFQ-EPR spectra and simulations of the protein-based radical signals in Mtb KatG

L-3,3-[²H]₂ tyrosine-labeled KatG (50 μM final) mixed with PAA (150 μM final) and frozen after 250 ms reaction at 25 °C (A). (The spectrum was taken from (10), Fig. 3e). Simulation of the tryptophanyl radical signal (wide doublet) (B). Simulation of the deuterium-substituted tyrosyl radical spectrum using adjusted hyperfine coupling parameters obtained from simulation of the doublet for unlabeled KatG (C). The simulated sum spectrum composed of 80% deuterium-substituted tyrosyl radical and 20% tryptophanyl radical signal intensities (D); g- tensor and hyperfine coupling values are given in Tables 1 and 2.

Fig. 6. RFQ-EPR spectra of WT KatG (solid lines) and KatG[W91F] mutant (dotted lines)

Resting enzymes (50 μM final) were mixed with peroxyacetic acid (150 μM final) at 25 °C. Reaction mixtures were freeze-quenched after (A) 50 ms; (B) 200 ms (wide doublet); (C) 10 s (wide singlet). The difference spectrum (dashed line) obtained by subtraction of the normalized experimental spectra of the wild type and KatG[W91F] is also shown.

Fig. 7. RFQ-EPR spectra of KatG[W107F][Y229F]

Resting enzyme (50 μM final) was mixed with PAA (150 μM final) and freeze-quenched after 250 ms (A). The WT KatG had been pre-treated with a 10-fold excess of PAA (B) (see text). g-values of peroxyl radical are indicated.