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 Fe-57(2+) show that Fe-57(3+) is the major species present under anaerobic conditions. This result suggests that the incoming Fe-57(2+) undergoes an internal redox reaction to form Fe-57(3+) during the formation of the P-i layer. Addition of bipyridine removes the Fe-57(3+) bound in the P-i layer as [Fe-57(bipy)(3)](2+), showing that the bound Fe-57(2+) has not undergone irreversible oxidation. This result is related to previous studies showing that Fe-57(2+) bound to native core is reversibly oxidized under anaerobic conditions in native hole bacterial and HoSF ferritins. Attempts to bury the P-i layer of native or reconstituted KoSF 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.