Forming the phosphate layer in reconstituted horse spleen ferritin and the role of phosphate in promoting core surface redox reactions

Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe³⁺/HoSF) by depositing Fe(OH)₃ within the hollow HoSF interior by air oxidation of Fe²⁺, Fe²⁺ and phosphate (P(i)) were then added anaerobically at a 1:4 ratio, and both Fe²⁺ and P(i) were incorporated into the HoSF cores. The resulting P(i) layer consisted of Fe²⁺ and P(i) at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe²⁺. The total amount of Fe²⁺ and P(i) bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the P(i) layer decreases with increasing core size. Mossbauer spectroscopic measurements of the P(i)- reconstituted HoSF cores using ⁵⁷Fe²⁺ show that ⁵⁷Fe³⁺ is the major species present under anaerobic conditions. This result suggests that the incoming ⁵⁷Fe²⁺ undergoes an internal redox reaction to form ⁵⁷Fe³⁺ during the formation of the (P)i layer. Addition of bipyridine removes the ⁵⁷Fe³⁺ bound in the P(i) layer as [⁵⁷Fe(bipy)₃]²⁺, showing that the bound ⁵⁷Fe²⁺ has not undergone irreversible oxidation. This result is related to previous studies showing that ⁵⁷Fe²⁺ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the P(i) layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the P(i) layer 'floats' on the developing iron mineral core.