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Link to original content: https://pubmed.ncbi.nlm.nih.gov/19139099
An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG) - PubMed Skip to main page content
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. 2009 Mar 13;284(11):7017-29.
doi: 10.1074/jbc.M808106200. Epub 2009 Jan 12.

An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG)

Javier Suarez  1 , Kalina Ranguelova, Andrzej A Jarzecki, Julia Manzerova, Vladimir Krymov, Xiangbo Zhao, Shengwei Yu, Leonid Metlitsky, Gary J Gerfen, Richard S Magliozzo
Affiliations

An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG)

Javier Suarez et al. J Biol Chem. .

Abstract

A mechanism accounting for the robust catalase activity in catalase-peroxidases (KatG) presents a new challenge in heme protein enzymology. In Mycobacterium tuberculosis, KatG is the sole catalase and is also responsible for peroxidative activation of isoniazid, an anti-tuberculosis pro-drug. Here, optical stopped-flow spectrophotometry, rapid freeze-quench EPR spectroscopy both at the X-band and at the D-band, and mutagenesis are used to identify catalase reaction intermediates in M. tuberculosis KatG. In the presence of millimolar H2O2 at neutral pH, oxyferrous heme is formed within milliseconds from ferric (resting) KatG, whereas at pH 8.5, low spin ferric heme is formed. Using rapid freeze-quench EPR at X-band under both of these conditions, a narrow doublet radical signal with an 11 G principal hyperfine splitting was detected within the first milliseconds of turnover. The radical and the unique heme intermediates persist in wild-type KatG only during the time course of turnover of excess H2O2 (1000-fold or more). Mutation of Met255, Tyr229, or Trp107, which have covalently linked side chains in a unique distal side adduct (MYW) in wild-type KatG, abolishes this radical and the catalase activity. The D-band EPR spectrum of the radical exhibits a rhombic g tensor with dual gx values (2.00550 and 2.00606) and unique gy (2.00344) and gz values (2.00186) similar to but not typical of native tyrosyl radicals. Density functional theory calculations based on a model of an MYW adduct radical built from x-ray coordinates predict experimentally observed hyperfine interactions and a shift in g values away from the native tyrosyl radical. A catalytic role for an MYW adduct radical in the catalase mechanism of KatG is proposed.

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Figures

FIGURE 1.
FIGURE 1.
The distal side structure of M. tuberculosis KatG (from the coordinates of PDB code 2CCA) showing the covalent Met-Tyr-Trp adduct.
FIGURE 2.
FIGURE 2.
RFQ-EPR of M. tuberculosis KatG reacted with H2O2. Top, samples were frozen at the indicated time points after mixing ferric KatG with 1000-fold excess H2O2 at pH 7.2 or 8.5. Spectra (average of nine scans) were recorded using the following conditions: T = 77 K, microwave frequency = 9.442 GHz; microwave power = 0.1 milliwatt; modulation amplitude = 1 G. Bottom, intensity (spins/heme) of the narrow doublet EPR signal as a function of time in RFQ-EPR samples frozen after mixing KatG with 1000-fold excess of H2O2 at pH 8.5. The curve is a fit of the data to a single exponential function using Sigma Plot 9.0.
FIGURE 3.
FIGURE 3.
Optical stopped-flow spectra of M. tuberculosis KatG reacted with H2O2. A, spectrum (solid line) recorded after 5 ms of incubation, an intermediate spectrum (dashed line) recorded 3.5 s after mixing, and a spectrum (dotted line) recorded 10 s after mixing KatG with 1000-fold excess of H2O2 at pH 6. B, time course of the reaction followed at 407 and 580 nm (pH 6). C, spectrum (solid line) recorded after 1.3 ms of incubation, an intermediate spectrum (dashed line) recorded 1.3 s after mixing, and a spectrum (dotted line) recorded 2 s after mixing KatG with 1000-fold excess of H2O2 at pH 8.5. D, time course of the reaction followed at 407 and 520 nm. These wavelengths correspond to the Soret maximum at the start of the time course and the new maximum in the visible region of the spectrum of the steady-state species formed at pH 8.5.
FIGURE 4.
FIGURE 4.
RFQ-EPR spectra of M. tuberculosis KatG reacted with 1000-fold molar excess of H2O2 frozen at the indicated time points. Spectra (average of 9 scans) were recorded under the following conditions: T = 4 K; microwave frequency = 9.3879 GHz; microwave power = 1 milliwatt; modulation amplitude = 4 G. At pH 8.5, a ferric heme iron intermediate is present (g = 2.24, 2.15, ?); At pH 7, the only signal present is the narrow doublet signal at g = 2.0034.
FIGURE 5.
FIGURE 5.
Manual freeze quench samples of KatG distal side mutants treated with 8000-fold molar excess of H2O2 at pH 8.5. Spectra (average of nine scans) were recorded under the following conditions: T = 77 K, microwave frequency = 9.442 GHz; microwave power = 0.1 milliwatt; modulation amplitude = 1 G.
FIGURE 6.
FIGURE 6.
A, echo-detected pseudo-modulated D-band (130 GHz) RFQ-EPR spectrum of M. tuberculosis KatG frozen 35 ms after mixing resting enzyme with 4000-fold molar excess of H2O2 at pH 8.5. Pulse widths, 40 and 80 ns; interpulse delay, 120 ns; temperature, 7 K; repetition rate, 10 Hz; averages per point, 30; 16 scans, total scan time 73 min. The overlaid simulation (dotted line) was generated as described in the text using the following parameters. Species 1 (59% of total simulation): gx = 2.00606, gy = 2.00344, gz = 2.00186, Ax = 11.9 G; Ay = 10.3 G; Az = 10.3 G. Species 2 (41% of total simulation) with same parameters as Species 1 except gx = 2.00550. Note that the smaller (∼2 G) hyperfine coupling required to fit the X-band simulation is unresolved because of inhomogeneous broadening in the D-band spectra. B, RFQ X-band EPR spectrum (solid line) of WT KatG treated with 1000-fold excess H2O2 at pH 8.5 frozen 20 ms after mixing. Experimental conditions, T = 77 K, microwave frequency = 9.442 GHz; microwave power = 0.1 milliwatt; modulation amplitude = 1 G; average of nine scans. Dotted line, simulation using the following parameters: g1,2,3 = 2.00606, 2.00344, 2.00186; Aiso(1) = 10.5 G, Aiso(2) = 3.2 G; line broadening = 3.6 G (average.). a.u., arbitrary units.
FIGURE 7.
FIGURE 7.
β-Methylene hydrogen orientation with respect to the unpaired electron π-orbital perpendicular to the phenol ring plane in a tyrosyl radical. The angle θ is defined for the strongly coupled hydrogen in an arrangement similar to that for Tyr229 in M. tuberculosis KatG.
FIGURE 8.
FIGURE 8.
A, environmental effect on computed isotropic Fermi coupling constant for the strongly couple hydrogen atom bound to the C-β carbon of Tyr229 in the MYW-O· adduct radical. B, DFT calculated unpaired spin density distribution (shown in blue) for the adduct using the polarizable continuum model (water) (isovalue = 0.012 e).
SCHEME 1.
SCHEME 1.
Proposed catalase reaction pathway.
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