Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies.
The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.
Saxena
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Chemical Physics Group, Tata Institute of Fundamental Research, Bombay, India.
Modi
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Behere
D V
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Mitra
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D006735:Horseradish Peroxidase / Q000378:metabolism*; D006859:Hydrogen; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism*; D010636:Phenols; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D010636:Phenols; D008345:Manganese; D006859:Hydrogen; D007501:Iron; D006735:Horseradish Peroxidase