Role of K+ binding residues in stabilization of heme spin state of Leishmania major peroxidase

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 H 2 O 2 . 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.