Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phoshate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe Mossbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a signigicant influence on iron depositon. Following oxidation, an intermediate mononuclear Fe-(III)-protein complex is formed which exhibits a transient EPR singal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. The mononuclear g' = 4.3 complex may arise from Fe(III) finding at a ferroxidase site or a nucleation site to which the Fe(III) has rapidly migrated following oxidation. Titration data suggest that a 3:1 Pi/Fe(III) complex is formed as the g' = 4.3 EPR signal decays. This complex may play a role in the migration of iron within the protein to form iron clusters, ultimately leading to formation of the core. The clusters can accommodate up to 0.38 ± 0.03 Pi/Fe as measured by equilibrium dialysis