The dioxygenation rate in lipoxygenase catalysis is determined by the amount of iron (III) lipoxygenase in solution.

The dioxygenation rate in reactions catalyzed by lipoxygenase-1 from soybeans has been measured as a function of the enzyme present in the Fe(III) form with rapid kinetic techniques. The experiments were carried out at pH 10, 25 degree C. The product concentration and the fraction of iron (III) lipoxygenase were monitored by measuring the absorbance at 243 nm and the tryptophan fluorescence at 330 nm (excitation at 287 nm), respectively. In reactions started with 1.3 microM iron (II) lipoxygenase and 9 microM linoleate, the initial rate, r(init) (estimated from the increase in absorbance over the initial 0.02 s of the reaction), is very small (4 s-1). In contrast, when the reactions are started with 1.3 microM (III) lipoxygenase, r(init) is large (150 s-1). In reactions started with mixtures of iron(II) and iron(III) lipoxygenase, r(init) is linearly related to the initial concentration of the Fe (III) enzyme form. Redistributions of the Fe(II) and Fe(III) enzyme forms during the reaction with 12 nM enzyme and 10, 50, or 100 microM linoleate appear to be directly reflected in changes in the dioxygenation rate. The observations provide solid evidence for the hypothesis that only iron (III) lipoxygenase can catalyze the hydrogen abstraction step in the dioxygenation reaction, and thus can be regarded as the active enzyme species. The observed dynamics are accurately predicted by a nonallosteric, two-step model for lipoxygenase catalysis [Schilstra et al. (1992) Biochemistry 31, 7692-7699].