An Oxyferrous Heme/Protein-based Radical Intermediate Is Catalytically Competent in the Catalase Reaction of Mycobacterium tuberculosis Catalase-Peroxidase (KatG)

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

doi:10.1074/jbc.m808106200

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.

Highlights

Unlike classical catalases which do not accumulate intermediates during H2O2 turnover because of very rapid rates of both the peroxide reduction and the peroxide oxidation steps, KatG forms a species characteristic of oxyferrous heme in the presence of high concentrations of peroxide [11, 15, 18]. This intermediate is usually stable in peroxidases and is considered to be catalytically inert, but it must be highly unstable in KatG because catalase turnover occurs while the heme is in this form (In cytochrome c peroxidase oxyferrous heme is unstable because of internal redox chemistry involving Trp191, but cytochrome c peroxidase does not exhibit catalase activity [19].) and the catalase activity of the W321F mutant of M. tuberculosis KatG is only moderately reduced [20]
RFQ-EPR and Optical Stopped-flow Experiments—The high catalase activity of M. tuberculosis KatG is conveniently measured in the presence of millimolar H2O2 without enzyme degradation or apparent inhibition
The presence of a tyrosyl-like radical in M. tuberculosis KatG during catalase turnover has been demonstrated by RFQ-EPR spectroscopy

Summary

Introduction

Unlike classical catalases which do not accumulate intermediates during H2O2 turnover because of very rapid rates of both the peroxide reduction and the peroxide oxidation steps, KatG forms a species characteristic of oxyferrous heme (peroxidase Cmpd III) in the presence of high concentrations of peroxide [11, 15, 18] This intermediate is usually stable in peroxidases and is considered to be catalytically inert, but it must be highly unstable in KatG because catalase turnover occurs while the heme is in this form (In cytochrome c peroxidase oxyferrous heme is unstable because of internal redox chemistry involving Trp191, but cytochrome c peroxidase does not exhibit catalase activity [19].) and the catalase activity of the W321F mutant of M. tuberculosis KatG is only moderately reduced [20]. A protein-based radical proposed here to be formed on the distal side amino acid adduct, which co-exists with oxyferrous heme when peroxide turnover occurs, is suggested to be a catalytically competent intermediate in catalase turnover in WT KatG

Methods

Results

Conclusion