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

Abstract Mycobacterium tuberculosis catalase-peroxidase ( Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous–CO complex of KatG(Y229F) suggest a non-linear Fe–CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met–Tyr–Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.