Hydrogen peroxide formation during iron deposition in horse spleen ferritin using O-2 as an oxidant

The reaction of Fe2+ with O-2 in the presence of horse spleen ferritin (HoS F) results in deposition of Fe(OH)(3) into the hollow interior of HoSF. Thi s reaction was examined at low Fe2+/HoSF ratios (5-100) under saturating ai r at pH 6.5-8.0 to determine if H2O2 is a product Of the iron deposition re action. Three methods specific for H2O2 detection were used to assess H2O2 formation: (1) a fluorometric method with emission at 590 nm, (2) an optica l absorbance method based on the reaction H2O2 + 3I(-) + 2H(+) = I-3(-) + 2 H(2)O monitored at 340 nm for I-3(-) formation, and (3) a differential puls ed electrochemical method that measures O-2 and H2O2 concentrations simulta neously. Detection limits of 0.25, 2.5, and 5.0 muM H2O2 were determined fo r the three methods, respectively. Under constant air-saturation conditions (20% O-2) and for a 5-100 Fe2+/HoSF ratio, Fe2+ was oxidized and the resul ting Fe3+ was deposited within HoSF but no H2O2 was detected as predicted b y the reaction 2Fe(2+) + O-2 + 6H(2)O = 2Fe(OH)(3) + H2O2 + 4H(+). Two othe r sets of conditions were also examined: one with excess but nonsaturating O-2 and another with limiting O-2. No H2O2 was detected in either case. The absence of H2O2 formation under these same conditions was confirmed by mic rocoulometric measurements. Taken together, the results show that under low iron loading conditions (5-100 Fe2+/HoSF ratio), H2O2 is not produced duri ng iron deposition into HoSF using O-2 as an oxidant. This conclusion is in consistent with previous, carefully conducted stoichiometric and kinetic me asurements [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965], predicting that H2O2 is a quantitative product of the iron deposition react ion with O-2 as an oxidant, even though it was not directly detected. Possi ble explanations for these conflicting results are considered.