Factors affecting the line-shape of the EPR signal of high-spin Fe(III) in soybean lipoxygenase-1

Abstract

The yellow form of soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12), obtained upon addition of one molar equivalent of acid (13--HPOD) to the native enzyme, shows a complex EPR signal around g 6 which results from contributions of different high-spin Fe(III) species with rhombic or axial symmetry. The signal cannot be attributed to different enzyme-product complexes because removal of the products or variation of the concentration of the products in the enzyme solution does not lead to an EPR spectrum characteristic for one particular species. Upon varying the pH of the enzyme solution from 7 to 11 changes in the line-shape are observed, but no distinct spectrum of either a rhombic or an axial form could be observed. The relative amounts of the different species visible in the signal around g 6 are strongly affected by cyanide, primary alcohols or 13--hydroxyoctadecadienoic acid (reduced 13-- HPOD). The presence of either of these substances causes a shift to an axial type of spectrum, t-Butanol and sodium dodecyl sulfate induce a shift towards a more rhombic line-shape. A shift to an axial type of spectrum is observed after storage of the enzyme in the yellow form at 4°C. Storage of the native form at 4°C also leads to changes which become apparent after oxidation in a similar axial type of spectrum. Reversion to the original, more rhombic, spectrum is possible by ammonium sulfate precipitation. It is concluded that the species giving rise to the EPR signal around g 6 are enzyme species differing only in the structure of the environment of the iron atom.