Divalent metal ion binding to the bacterial iron-storage protein, bacterioferritin (BFR), which contains a dinuclear metal binding site within each of its 24 subunits, was investigated by potentiometric and spectrophotometric methods. Cobalt(II) and zinc(II) were found to bind at both high- and low-affinity sites. Cobalt(II) binding at the high-affinity site was observed at a level of two per subunit with the release of ∼ 1.6 protons per metal ion, thus confirming the dinuclear metal centre as the high-affinity site. Zinc(II) binding at the dinuclear centre (high-affinity site) resulted in the release of ∼ 2 protons per metal ion, but exhibited a binding stoichiometry which indicated that not all dinuclear centres were capable of binding two zinc(II) ions. Competition data showed that binding affinities for the dinuclear centre were in the order zinc(II) > cobalt(II), and also confirmed the unexpected stoichiometry of zinc(II) binding. This work emphasises the importance ∼ of charge neutrality at the dinuclear centre.
