Analysis of Heme Structural Heterogeneity in Mycobacterium tuberculosis Catalase-Peroxidase (KatG)

Abstract

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 sixcoordinate 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.

The Met-Tyr-Trp Cross-link in Mycobacterium tuberculosis Catalase-peroxidase (KatG)

“…Thus, as can be seen from the data in Table I, WT KatG has a greater amount of 5-c HS heme present than KatG(Y229F), and both lack a detectable (by optical spectroscopy) low spin heme component. These results are consistent with previous observations for these two enzymes (47,48).…”

“…As described elegantly by Magliozzo and co-workers (48), analysis of the two ratios A Soret /A 380 and A 614 /A 645 reveals the relative populations of 6-c versus 5-c HS heme: overall, 5-c HS heme species exhibit a slightly blue shifted and lower extinction coefficient Soret band than their 6-c HS counterparts, as well as a shoulder at 380 nm. Additionally, the CT1 feature in a 5-c HS heme is found at ϳ640 nm (or higher), whereas that of a 6-c HS heme is generally closer to 630 nm.…”

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met–Tyr–Trp cross-linked adduct

“…In KatG(Y229F), a significant amount of LS heme is present that appears to come largely at the expense of the QS heme contribution when compared to WT KatG, which does not have detectable levels of LS heme [33,34]. The contributions of the four different heme species in KatG(Y229F) are rather insensitive to pH, though a small increase in LS heme is observed with increasing pH at the expense of the other heme species.…”

“…The plasmid pKATII was used for site-directed mutagenesis to produce KatG(Y229F) as described previously [20,32,33]. The mutant was overexpressed in E. coli strain UM262 (KatG minus) expressing the M. tuberculosis katG gene provided by Stewart Cole (Institute Pasteur, Paris).…”

“…The bacteria were grown in the presence of the heme biosynthetic precursor, δ-aminolevulinic acid. KatG(Y229F) was isolated and purified according to published procedures [32][33][34]. Purified enzyme was stored in 20 mM potassium phosphate buffer at pH 7.2.…”