Spectroscopic comparison of the heme active sites in WT KatG and its S315Tmutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been cha racterized by resonance Raman, electron spin resonance, and visible spectro scopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-r esistant clinical isolates, is also spectroscopically characterized. The el ectron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 24 4 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolat e character of the proximal histidine in their five-coordinate high-spin co mplexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures o f six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a maj ority of the heme exists in a live-coordinate high-spin state, but six-coor dinate hemes are also present. At room temperature, more six-coordinate low -spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enz yme. While the nature of the sixth ligand of LS ferric wild-type KatG is no t completely clear, visible, resonance Raman, and electron spin resonance d ata of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen don or. Possible effects of these differences on enzyme activity are discussed.