Kinetics and mechanism of iron oxidation in apoferritin

Abstract

The enzymatic activity of horse spleen apoferritin in iron(II) oxidation was examined using microelectrode oximetry. The reaction exhibits saturation kinetics with respect to both Fe\sp{2+} and O\sb2. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe\sp{2+} oxidation occurs in 1-electron steps. At increments of 50 Fe\sp{2+}/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. The results of these studies emphasize the role of the protein shell in all phases of core growth. A detailed study of the kinetics of iron(II) oxidation by molecular oxygen in natural and recombinant human apoferritins has also been carried out to understand the ferroxidase activity of the protein shell and the function of the H and L subunits during iron uptake in ferritins. Zn\sp{2+} was shown to be a noncompetitive inhibitor of Fe\sp{2+} oxidation in rHF but a mixed inhibitor in HLF. These different forms of Zn\sp{2+} inhibition in the two proteins and the higher than expected activity of HLF based on its H-chain composition as well as differences in their enzyme kinetic parameters suggest that H and L-chains cooperate in modulating the ferroxidase activity of the apoferritin even though the L-subunit lacks a ferroxidase site itself. Additionally, the intermediate species produced in the process of ferritin reconstitution from apoferritin, Fe\sp{2+} and dioxygen, have been investigated using fast-mixing and stir-mixing freeze-quench techniques and EPR spectroscopy. The intermediate species found include the previously defined monomeric Fe\sp{3+}-protein complex (g\sp\prime = 4.3), the mixed-valence Fe\sp{2+}-Fe\sp{3+} intermediate (g\sp\prime = 1.87) and the free radical, as well as a new radical with axial magnetic symmetry. Interaction of Fe\sp{2+} with the monomeric Fe\sp{3+}-protein complex was demonstrated. A 1:1 relationship between the monomeric Fe\sp{3+}-protein complex and the mixed-valence species was observed within the first second of reaction. The temperature-dependent properties of the new radical suggest that it may be associated with an iron center and may be a tryptophan-centered radical in the \rm Fe\sp{2+}/Fe\sp{3+}/apoprotein system