CHARACTERIZATION OF THE NONHEME IRON CENTER OF HUMAN 5-LIPOXYGENASE BY ELECTRON-PARAMAGNETIC-RESONANCE, FLUORESCENCE, AND ULTRAVIOLET-VISIBLE SPECTROSCOPY - REDOX CYCLING BETWEEN FERROUS AND FERRIC STATES

Purified human 5-lipoxygenase, a non-heme iron containing enzyme, has been characterized by electron paramagnetic resonance, (EPR), ultraviolet (UV)-visible and fluorescence spectroscopy. As isolated, the enzyme is largely in the ferrous state and shows a weak X-band EPR signal extending from 0 to 700 G at 15 K, tentatively ascribed to integer spin Fe(II). Titration of the protein with 13-HPOD (13-hydroperoxyoctadecadienoic acid) generates a strong multicomponent EPR signal in the g' almost-equal-to 6 region, a yellow color associated with an increased absorption between 310 and 450 nm (epsilon330nm = 2400 M-1 cm-1), and a 17% decrease in the intrinsic protein fluorescence. The multiple component nature of the g' almost-equal-to 6 signal indicates that the metal center in its oxidized state exists in more than one but related forms. The g' almost-equal-to 6 EPR signal and the yellow color reach a maximum when approximately 1 mol of 13-HPOD is added/mol of iron; the resultant EPR spectrum accounts quantitatively for all of the iron in the protein with a signal at g' = 4.3 representing less than 3% of the total iron in the majority of samples. Addition of a hydroxyurea reducing agent abolished the g' almost-equal-to 6 signal and yellow color of the protein and also reversed the decrease in fluorescence caused by the oxidant 13-HPOD. The results indicate that the g' almost-equal-to 6 EPR signal, the yellow color, and the decreased fluorescence are associated with the formation of the Fe(III) form of the enzyme. The spectroscopic data demonstrate that the Fe(III) center of 5-lipoxygenase can be redox-cycled, consistent with its postulated role in the mechanism of oxidation of unsaturated fatty acids to hydroperoxides. Studies using the irreversible inhibitor 4-nitrocatechol, which binds to the ferric form of the enzyme, are also consistent with the redox cycling of the iron and demonstrate that the oxidation state of the enzyme influences both its functional and physical properties.