Superoxide ion as a primary reductant in ascorbate-mediated ferretin iron release

Abstract

The mechanism of ascorbate-promoted ferritin iron reduction under aerobic conditions was studied. The unitial rate of ferritin iron release was determined by spectrophotometric measurement of the Fe(ferrozine) 3 2+ complex which absorbs at 562 nm. Variation of the initial ferrozine concentration had no influence on the rate of iron release suggesting that ferrozine does not participate in the rate-determining step. Experimental measurements of the initial rate of iron release as a function of ascorbate concentration resulted in saturation kinetics with V max = 2.0 × 10 −7 M · min −1 and K M = 1.3 × 10 −3 M. The effect of pH was quite pronounced with a maximal rate of iron release at pH 7.0. Stoichiometric measurements on the reaction mixture, with added catalase, resulted in a ratio of 2 Fe(II) released per ascorbate. Ascorbate-mediated iron release was inhibited 85% by superoxide dismutase, but 0% inhibition was noted with aposuperoxide dismutase. It is proposed that superoxide ion, generated during the iron-promoted oxidation of ascorbate, acts as a reductant of ferritin iron. A mechanism of ferritin iron release consistent with these experimental observations is discussed.