Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG).
Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme. Five-coordinate heme is suggested to be representative of "native" enzyme, since this species was predominant in the enzyme examined immediately after one chromatographic protocol. Quantum mechanically mixed spin heme is the most abundant form in such partially purified enzyme. Reduction and reoxidation of six-coordinate KatG or the addition of glycerol or isoniazid restored five-coordinate heme iron, consistent with displacement of a weakly bound distal water molecule. The rate of formation of KatG Compound I is not retarded by the presence of six-coordinate heme either in wild-type KatG or in a mutant (KatG[Y155S]) associated with isoniazid resistance, which contains abundant six-coordinate heme. These results reveal a number of similarities and differences between KatG and other Class I peroxidases.
Chouchane
Salem
S
Department of Chemistry, Brooklyn College and the Graduate Center of the City University of New York, 11210-2889, USA.
Girotto
Stefania
S
Kapetanaki
Sofia
S
Schelvis
Johannes P M
JP
Yu
Shengwei
S
Magliozzo
Richard S
RS
D001426:Bacterial Proteins; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry*; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D006418:Heme; D010544:Peroxidases; C076136:catalase HPI