Application of EPR spectroscopy to study the resting state structure and the mechanism of Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis (M. tuberculosis ) catalase-peroxidase (KatG) is a dimeric Class I heme peroxidase whose activity is implicated for the activation of the anti-tuberculosis antibiotic isoniazid (INH).; The catalytic function and the structure of this enzyme have been examined using rapid freeze-quench (RFQ and low temperature X-band EPR spectroscopy.; The enzyme exhibits catalytic properties that differ from the Class I peroxidases. The reaction of ferric KatG with peroxyacetic acid was followed using RFQ-EPR (77 K). A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s. Simulation of EPR spectra and isotope labeling experiments assigned both doublet and singlet EPR signals to tyrosyl radical(s). A two-state model was found to be adequate to describe the kinetics of evolution of the EPR signal from doublet to singlet observed in X-band data while High Field EPR results suggest that a distribution of orientations are present.; Single amino acid replacements in KatG have been investigated as a direct approach to identify the tyrosine residues at which the radical(s) is formed. RFQ-EPR spectroscopy confirms that tyrosine Y353, unique to M. tuberculosis KatG, is the amino acid at which a tyrosyl radical is formed upon turnover with peroxides. Moreover, residue Y229, which is involved in the formation of a newly defined Met-Tyr-Trp adduct in the active site of catalase-peroxidase, is shown to be important for preserving the catalase activity of KatG.; Low temperature EPR studies of ferric KatG, supported by optical and Raman data, suggest that different factors, such as water and other ligand binding, protonation state of the distal imidazole and incomplete adduct formation, are responsible for the heme iron structural heterogeneity observed in the WT enzyme. Coordination of the ferric iron and the geometry of the active site are influenced by small molecules such as INH, which binds at a special binding site removed from the heme. Moreover, alterations in the region of Ser315, whether induced by mutation or INH binding, affect the hydrogen bonding network on the distal side of the heme.