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.

Impact of Phosphate on Iron Mineralization and Mobilization in Nonheme Bacterioferritin B from Mycobacterium tuberculosis

“…Iron oxidation and mineralization reactions occur differently in different types of ferritins, where H 2 O 2 may act as a cosubstrate or as a byproduct of the ferroxidase reaction and hence contributes to Fe 2+ /O 2 stoichiometry. 10,24,53,60 In order to check whether H 2 O 2 is generated or not during the ferroxidase reaction of Mtb BfrB, both catalase (monitoring the evolution of O 2 using oximetry) and peroxidase (monitoring the formation of resorufin using spectrophotometry) assays were performed as reported earlier. 10,24,53 The evolution of O 2 upon catalase treatment indicates the generation of H 2 O 2 during the ferroxidase reaction of Mtb BfrB (Figure 4C).…”

“…In addition, it stabilizes Fe 3+ more than Fe 2+ (decrease in the redox potential by 600 mV), 67 owing to its strong complexation with Fe 3+ at neutral pH (Binding affinity, K > 10 8 M −1 ). 10,53,60 Hence, phosphate not only facilitates the iron oxidation in buffer but also competes with the F ox center and alters the kinetics and mechanism of iron oxidation/mineralization inside the ferritin protein nanocage (Figure 3). The impact of phosphate during ferroxidase activity (phase 2) was found to be significant at higher iron loading (480 Fe/cage).…”

Iron Mineralizing Bacterioferritin A from Mycobacterium tuberculosis Exhibits Unique Catalase-Dps-like Dual Activities

“…The kinetics of dissolved O 2 consumption during the ferroxidase reaction of Mtb BfrA further revealed the participation of both H 2 O 2 and O 2 as Fe 2+ oxidant. The variation obtained in the Fe 2+ /O 2 stoichiometry (Figure S13) during the single and multiple additions of FeSO 4 to Mtb BfrA can be attributed to several factors such as the effects of catalase activity (a new finding in the current report), Fe 2+ loading per cage, MOPS buffer, etc., which significantly affects the Fe 2+ /O 2 stoichiometry during the ferroxidase activity of ferritin. − Similar to eukaryotic ferritin, high iron loading (∼500 Fe/Cage) leads to complete reduction of O 2 to H 2 O with the stoichiometry being very close to the expected value of 4:1 (Figure C). However, with 120 Fe/Cage, the ratio (2.0 ± 0.1) was close to the value (∼2:1) since the H 2 O 2 generated via the oxidation of 2 mol of Fe 2+ by 1 mol of O 2 further gets possibly consumed by MOPS buffer as well as by catalase-like activity of Mtb BfrA, thus preventing it from generating the desired stoichiometry of 4:1 (Figure D and Figure S13).…”

Anaerobic iron deposition into horse spleen, recombinant human heavy and light and bacteria ferritins by large oxidants

“…The complications arise because O 2 is reduced to H 2 O 2 , which either reacts with Fe(II) to form mineral core (Fe(II)/O 2 = 4.0) or undergoes secondary reactions with solution components (Fe(II)/O 2 = $2.5). The sites of the secondary reactions were initially considered to be the protein shell or the mineral core [14], but subsequent results show that buffer oxidation by H 2 O 2 is the dominant reaction [12,15]. Thus, while the use of O 2 is convenient for iron deposition studies, the resulting side reactions complicate mechanistic studies and it is desirable to investigate the reactivity of other oxidants.…”