title: Role of the oxyferrous heme intermediate and distal side adduct radical in the catalase activity of Mycobacterium tuberculosis KatG revealed by the W107F mutant.
abstract: Catalase-peroxidase (KatG) is essential in Mycobacterium tuberculosis for oxidative stress management and activation of the antitubercular pro-drug isoniazid. The role of a unique distal side adduct found in KatG enzymes, involving linked side chains of residues Met255, Tyr229, and Trp107 (MYW), in the unusual catalase activity of KatG is addressed here and in our companion paper (Suarez, J., Ranguelova, K., Jarzecki, A. A., Manzerova, J., Krymov, V., Zhao, X., Yu, S., Metlitsky, L., Gerfen, G. J., and Magliozzo, R. S. (2009) J. Biol. Chem. 284, in press). The KatG[W107F] mutant exhibited severely reduced catalase activity yet normal peroxidase activity, and as isolated contains more abundant 6-coordinate heme in high spin and low spin forms compared with the wild-type enzyme. Most interestingly, oxyferrous heme is also found in the purified enzyme. Oxyferrous KatG[W107F] was prepared by photolysis in air of the carbonyl enzyme or was generated using hydrogen peroxide decayed with a t1/2 of 2 days compared with 6 min for wild-type protein. The stability of oxyenyzme was modestly enhanced in KatG[Y229F] but was not affected in KatG[M255A]. Optical stopped-flow experiments showed rapid formation of Compound I in KatG[W107F] and facile formation of oxyferrous heme in the presence of micromolar hydrogen peroxide. An analysis of the relationships between catalase activity, stability of oxyferrous enzyme, and a proposed MYW adduct radical is presented. The loss of catalase function is assigned to the loss of the MYW adduct radical and structural changes that lead to greatly enhanced stability of oxyenzyme, an intermediate of the catalase cycle of native enzyme.
authors.affiliation: Department of Chemistry, Brooklyn College, Brooklyn, New York 11210, USA.
authors.forename: Xiangbo
authors.initials: X
authors.lastname: Zhao
authors.forename: Shengwei
authors.initials: S
authors.lastname: Yu
authors.forename: Kalina
authors.initials: K
authors.lastname: Ranguelova
authors.forename: Javier
authors.initials: J
authors.lastname: Suarez
authors.forename: Leonid
authors.initials: L
authors.lastname: Metlitsky
authors.forename: Johannes P M
authors.initials: JP
authors.lastname: Schelvis
authors.forename: Richard S
authors.initials: RS
authors.lastname: Magliozzo
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002374:Catalase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism; D007538:Isoniazid / Q000737:chemistry; D008956:Models, Chemical; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D011355:Prodrugs / Q000737:chemistry; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011355:Prodrugs; D006418:Heme; D006861:Hydrogen Peroxide; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid