Iron extraction from soybean lipoxygenase 3 and reconstitution of catalytic activity from the apoenzyme

Lipoxygenases contain a unique nonheme iron cofactor with a redox role in t he catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal o f the iron by o-phenanthroline was attained in the presence of substrate un der anaerobic conditions, but the apoenzyme could not be isolated and recon stituted. The freshly regenerated sodium form of Chelex-100 also removes th e iron atom from native soybean lipoxygenase 3, but only in sodium bicarbon ate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris-HCl buffer at pH 7.0. Stoichiometric i ron in the reconstituted enzyme was established using inductively coupled p lasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 /- 10% of the specific activity of the native enzyme. The native configurat ion of the reconstituted iron site was confirmed by electron paramagnetic r esonance spectroscopy. (C) 2001 Academic Press.