Characterization of non-heme iron center in potato tuber lipoxygenase by EPR spectroscopy
Butovich, I.A.; Pechous, S.; Reddy, C.C.anna
FASEB Journal 15(5): A876
Lipoxygenases (LOXs, EC 184.108.40.206) are a family of closely related non-heme iron enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids (PUFAs) containing one or more 1,4-pentadiene moieties. In the resting state, all the known LOXs contain one essential iron atom in Fe (II)-form at the active center. The Fe(II)/Fe(III)-cycling is the essential part of its catalytic mechanism. Determination of the oxidation state of the iron is critical for understanding the kinetics and reaction mechanism of the LOX-catalyzed reactions. We report here the results of our EPR spectroscopy data on the electrophoretically pure LOX-2 from potato tubers (S. tuberosum L.). An EPR spectrum of this LOX-2 recorded at apprx4-5 K showed strong signals at g values of 6.27 and 4.37. The first signal was attributed to catalytically relevant high spin non-heme Fe(III), which is characteristic of the "activated" LOX. The second weaker signal with g-value of apprx4, known as "rhombic" iron, belongs to hexacoordinated Fe (III) and is attributed to catalytically irrelevant iron. When the "native" LOX-2 was treated with apprx6x molar excess of 9(S)-hydroperoxyoctadecadienoic acid (9S-HPODE), a substantial (apprx3-fold) increase in the intensity of the signal at g value of apprx6.3 was observed. Based on these data, we conclude that approximately 25% of the "native" enzyme was in the EPR-visible Fe(III) form with the remaining enzyme in the EPR-silent Fe(II) state. The intensity of the signal at g value apprx4.4 also rose; however, it was not nearly as much as that of the major signal. Also, We have observed that 13(S)-HPODE, a positional isomer of 9(S)-HPODE, oxidized Fe(II) into Fe(III) to the same extent. Interestingly, however, 13(S)-HPODE treatment of native LOX-2 led to a distinct shoulder with a g value of 5.9, which is indicative of changes in the coordination sphere of non-heme iron.