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J. Biol. Chem., Vol. 278, Issue 45, 44121-44127, November 7, 2003

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Rapid Formation of Compound II and a Tyrosyl Radical in the Y229F Mutant of Mycobacterium tuberculosis Catalase-peroxidase Disrupts Catalase but Not Peroxidase Function^*

Shengwei Yu, Stefania Girotto, Xiangbo Zhao, and Richard S. Magliozzo

From the Departments of Chemistry and Biochemistry, Brooklyn College and the Graduate Center of the City University of New York, Brooklyn, New York 11210

Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme -cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.

Received for publication, May 7, 2003 , and in revised form, July 18, 2003.

^* This work was supported by NIAID/National Institutes of Health Grant AI-43582 (to R. S. M.) and the Heiser Program for Research in Leprosy and Tuberculosis (to S. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, Brooklyn College, 2900 Bedford Ave., Brooklyn, NY 11210. Tel.: 718-951-4174; Fax: 718-951-4607; E-mail: rmaglioz{at}brooklyn.cuny.edu.

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