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

• Published in: The Journal of biological chemistry Vol. 284; no. 11; pp. 7017 - 7029
• Main Authors: Suarez, Javier, Ranguelova, Kalina, Jarzecki, Andrzej A., Manzerova, Julia, Krymov, Vladimir, Zhao, Xiangbo, Yu, Shengwei, Metlitsky, Leonid, Gerfen, Gary J., Magliozzo, Richard S.
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 13.03.2009
• Subjects: Enzyme Catalysis and Regulation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M808106200

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 H 2 O 2 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 H 2 O 2 (1000-fold or more). Mutation of Met 255 , Tyr 229 , or Trp 107 , 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 g x values (2.00550 and 2.00606) and unique g y (2.00344) and g z 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.