Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.
The kinetics of E. coli bacterioferritin di‐ferrous ferroxidase centre reacting with O2 and H2O2 shows that H2O2 reacts circa 1000 times faster than O2 implying that the primary in vivo role of the protein is ROS detoxification rather than iron sequestering.
Keywords: EPR spectroscopy; Mössbauer spectroscopy; fast kinetics; ferroxidase center; rapid freeze-quenching.
© 2021 The Authors. Angewandte Chemie published by Wiley-VCH GmbH.