The Iron Oxidation and Hydrolysis Chemistry of Escherichia coli Bacterioferritin

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Abstract
Bacterioferritins are members of a class of spherical shell-like iron storage proteins that catalyze the oxidation and hydrolysis of iron at specific sites inside the protein shell, resulting in formation of a mineral core of hydrated ferric oxide within the protein cavity. Electrode oximetry/pH stat was used to study iron oxidation and hydrolysis chemistry in E. coli bacterioferritin. Consistent with previous UV−visible absorbance measurements, three distinct kinetic phases were detected, and the stoichiometric equations corresponding to each have been determined. The rapid phase 1 reaction corresponds to pairwise binding of 2 Fe2+ ions at a dinuclear site, called the ferroxidase site, located within each of the 24 subunits, viz., 2Fe2+ + PZ → [Fe2−P]Z + 4H+, where PZ is the apoprotein of net charge Z and [Fe2−P]Z represents a diferrous ferroxidase complex. The slower phase 2 reaction corresponds to the oxidation of this complex by molecular oxygen according to the net equation: [Fe2−P]Z + 1/2O2 → [Fe2O−P]Z where [Fe2O−P]Z represents an oxidized diferric ferroxidase complex, probably a μ-oxo-bridged species as suggested by UV−visible and EPR spectrometric titration data. The third phase corresponds to mineral core formation according to the net reaction: 4Fe2+ + O2 + 6H2O → 4FeO(OH)(core) + 8H+. Iron oxidation is inhibited by the presence of Zn2+ ions. The patterns of phase 2 and phase 3 inhibition are different, though inhibition of both phases is complete at 48 Zn2+per 24mer, i.e., 2 Zn2+ per ferroxidase center.