Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy.

Sofia Kapetanaki, Salem Chouchane, Stefania Girotto, Shengwei Yu, Richard S Magliozzo, Johannes P M Schelvis

Department of Chemistry, New York University, 31 Washington Place, Room 1001, New York, New York 10003, USA.

Biochemistry 2003 Apr 8

filter terms:

KatG from Mycobacterium tuberculosis is a heme-containing catalase-peroxidase, which belongs to the class I peroxidases and is important for activation of the prodrug isoniazid (INH), a front-line antituberculosis drug. In many clinical isolates, resistance to INH has been linked to mutations on the katG gene, and the most prevalent mutation, S315T, suggests that modification of the heme pocket has occurred. Electronic absorption and resonance Raman spectra of ferric wild-type (WT) KatG and its INH-resistant mutant KatG(S315T) at different pH values and their complexes with INH and benzohydroxamic acid (BHA) are reported. At neutral pH, a quantum mechanically mixed spin state (QS) is revealed, which coexists with five-coordinate and six-coordinate high-spin hemes in WT KatG. The QS heme is the major species in KatG(S315T). Addition of either INH or BHA to KatG induces only minor changes in the resonance Raman spectra, indicating that both compounds do not directly interact with the heme iron. New vibrational modes are observed at 430, 473, and 521 cm(-1), and these modes are indicative of a change in conformation in the KatG heme pocket. The intensity of these modes and the relative population of the QS heme are stable in KatG(S315T) but not in the WT enzyme. This indicates that there are differences in heme pocket stability between WT KatG and KatG(S315T). We will discuss the stabilization of the QS heme and propose a model for the inhibition of INH oxidation by KatG(S315T).

Citation

Sofia Kapetanaki, Salem Chouchane, Stefania Girotto, Shengwei Yu, Richard S Magliozzo, Johannes P M Schelvis. Conformational differences in Mycobacterium tuberculosis catalase-peroxidase KatG and its S315T mutant revealed by resonance Raman spectroscopy. Biochemistry. 2003 Apr 8;42(13):3835-45