title: Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH.
abstract: Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the 'rhombic' fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the 'axial' form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.
authors.affiliation: School of Biological Science, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom. svist@essex.ac.uk
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
authors.forename: Snehpriya B
authors.initials: SB
authors.lastname: Chugh
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Haigh
authors.forename: Reza A
authors.initials: RA
authors.lastname: Ghiladi
authors.forename: Peter
authors.initials: P
authors.lastname: Nicholls
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002374:Catalase / Q000737:chemistry*; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D005290:Ferric Compounds; D006418:Heme; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis