A spin label study of horseradish peroxidase

Abstract

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid ( N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)- p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe 3+ or Mn 3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant K a ≈ 4.7 · 10 5 M −1. Since the interaction of ligands (e.g. F −, CN −) and H 2O 2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the p K value of the acid-alkali transition of horse-radish peroxidase was found to be 10.5. The 2 T m value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0° C and 46.5 G at 52° C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label