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

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

“…This species lacks the CT bands of the resting (ferric) enzyme at 500 and 630 -640 nm and has the features of oxyferrous enzyme, including a Soret maximum at 413 nm and with ␤ and ␣ bands at 545 and 579 nm. The spectrum of oxyferrous heme in M. tuberculosis KatG (11,15) was confirmed in the companion paper (69). The reaction pathway to oxyenzyme can occur through reduction of Fe(IV) by peroxide, superoxide addition to ferric heme, or dioxygen binding to ferrous iron.…”

“…The role of Arg 418 in modulating catalytic functions of KatG as a function of pH has also been addressed elsewhere without including the possibility of an MYW adduct radical (37). The results presented here and in the companion paper (69) suggest that a reexamination of the structure of intermediates in catalytic functions of KatG is warranted.…”

“…It is important to note that KatG[Y229F], W107F, and M255A mutants form oxyenzyme in the presence of excess hydrogen peroxide just as the WT enzyme does, which means that it is not loss of this heme intermediate that is responsible for the loss in catalase function. Furthermore, greatly enhanced stability of the oxyenzyme contributes to the lost catalase function in the KatG[Y229F] and W107F mutants (69).…”

“…and the catalase activity of the W321F mutant of M. tuberculosis KatG is only moderately reduced (20). In the companion paper (69), formation of oxyenzyme from the carbonyl enzyme shows that in WT KatG, oxyferrous heme is too stable to be a viable catalase intermediate unless some other unique feature produced in the oxyferrous enzyme formed in the presence of hydrogen peroxide alters its properties. 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.…”

Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG

“…These findings demonstrate that the MYW-adduct enables formation of a unique intermediate required for efficient turnover of H 2 O 2 . Using rapid freeze-quench (RFQ) EPR, we reported the characterization of a narrow doublet radical signal (17-Gauss linewidth, a H1,H2 ϭ 11 and 2.5 Gauss for ␤-methylene hydrogens) from a modified tyrosine in M. tb KatG (13)(14). More recent isotope labeling and mutagenesis experiments (15), allowed assignment of the radical found during catalase turnover to the MYW-adduct and elucidated a function for this unique cofactor first suggested several years ago (16).…”