Isolation and spectroscopic characterization of a recombinant bell pepper hydroperoxide lyase

Fatty acid hydroperoxide (HPO) lyase is a component of the oxylipin pathway and holds a central role in elicited plant defense. HPO lyase from bell pe pper has been identified as a heme protein which shares 40% homology with a llene oxide synthase, a cytochrome P450 (CYP74A). HPO lyase of immature bel l pepper fruits was expressed in Escherichia coli and the enzyme was purifi ed and characterized by spectroscopic techniques. The electronic structure and ligand coordination properties of the heme were investigated by using a series of exogenous ligands. The various complexes were characterized by u sing UV-visible absorption and electron paramagnetic resonance spectroscopy . The spectroscopic data demonstrated that the isolated recombinant HPO lya se has a pentacoordinate, high-spin heme with thiolate ligation. Addition o f the neutral ligand imidazole or the anionic ligand cyanide results in the formation of hexacoordinate adducts that retain thiolate ligation. The str iking similarities between both the ferric and ferrous HPO lyase-NO complex es with the analogous P450 complexes, suggest that the active sites of HPO lyase and P450 share common structural features. (C) 2001 Elsevier Science BN. All rights reserved.