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

作者：Suarez Javier; Ranguelova Kalina; Jarzecki Andrzej A; Manzerova Julia; Krymov Vladimir; Zhao Xiangbo; Yu Shengwei; Metlitsky Leonid; Gerfen Gary J; Magliozzo Richard S^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2009, 284(11): 7017-7029.

DOI：10.1074/jbc.M808106200

Amechanism 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.

• 出版日期2009-3-13