Role of K binding residues in stabilization of heme spin state of Leishmania major peroxidase |
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Authors: | Swati Pal Rajesh K Yadav Subrata Adak |
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Institution: | Division of Structural Biology and Bio-informatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700 032, India |
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Abstract: | The endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at 25 °C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin state of the heme iron, in favor of the enzymatically active form of LmP. |
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Keywords: | APX plant ascorbate peroxidase LmP Leishmania major peroxidase CCP yeast cytochrome c peroxidase HS high spin LS low spin WT wild type |
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