Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe²⁺ to O₂ stoichiometry of iron deposition in horse spleen ferritin

Abstract

Ferritin catalyzes the oxidation of Fe²⁺ by O₂ to form a reconstituted Fe³⁺ oxy-hydroxide mineral core, but extensive studies have shown that the Fe²⁺ to O₂ stoichiometry changes with experimental conditions. At Fe²⁺ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe²⁺ to O₂ of 4 is typically measured, indicating O₂ is reduced to 2H₂O. In contrast, a lower limit of Fe²⁺ to O₂ of approximately  2 is measured at low Fe²⁺ to HoSF ratios, implicating H₂O₂ as a product of Fe²⁺ deposition. Stoichiometric amounts of H₂O₂ have not been measured, and H₂O₂ is proposed to react with an unknown system component. Evidence is presented that identifies this component as amine buffers, including 3-N-morpholinopropanesulfonic acid (MOPS), which is widely used in ferritin studies. In the presence of non-amine buffers, the Fe²⁺ to O₂ stoichiometry was approximately  4.0, but at high concentrations of amine buffers (0.10 M) the Fe²⁺ to O₂ stoichiometry is approximately 2.5 for iron loadings of eight to 30 Fe²⁺ per HoSF. Decreasing the concentration of amine buffer to zero resulted in an Fe²⁺ to O₂ stoichiometry of approximately 4. Direct evidence for amine buffer modification during Fe²⁺ deposition was obtained by comparing authentic and modified buffers using mass spectrometry, NMR, and thin layer chromatography. Tris(hydroxymethyl)aminomethane, MOPS, and N-methylmorpholine (a MOPS analog) were all rapidly chemically modified during Fe²⁺ deposition to form N-oxides. Under identical conditions no modification was detected when amine buffer, H₂O₂, and O₂ were combined with Fe²⁺ or ferritin separately. Thus, a short-lived ferritin intermediate is required for buffer modification by H₂O₂. Variation of the Fe²⁺ to O₂ stoichiometry versus the Fe²⁺ to HoSF ratio and the amine buffer concentration are consistent with buffer modification.