Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant.
KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate 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 enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.
Lukat-Rodgers
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Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105, USA. rodgers@plains.nodak.edu.
Wengenack
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NL
Rusnak
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F
Rodgers
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KR
D001426:Bacterial Proteins; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008024:Ligands; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000235:genetics; D013059:Spectrum Analysis, Raman; D013912:Threonine / Q000235:genetics
D001426:Bacterial Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D013912:Threonine; D006418:Heme; D012694:Serine; D006639:Histidine; D010544:Peroxidases; C076136:catalase HPI