Iron environment in ferritin with large amounts of phosphate, from Azotobacter vinelandii and horse spleen, analyzed using extended X-ray absorption fine structure (EXAFS).

The iron core of proteins in the ferritin family displays structural variations that include phosphate content as well as the number and the degree of ordering of the iron atoms. Earlier studies had shown that ferritin iron cores naturally high in phosphate, e.g., Azotobacter vinelandii (AV) ferritin (Fe:P ratio = 1:1.7), had decreased long-range order. Here, the influence of phosphate on the local structure around iron in ferritin cores is reported, comparing the EXAFS of AV ferritin, reconstituted ferritin [the protein coats of horse spleen ferritin mixed with Fe(II) with and without phosphate at pH 7] (Fe:P ratio = 1:0.25), and native horse spleen ferritin (Fe:P ratio = 1:0.125); reconstituted horse spleen ferritin without phosphate was indistinguishable from native horse spleen ferritin (HSF) in the analysis. In contrast, when the phosphate content was high in AV ferritin and horse spleen ferritin reconstituted with phosphate, the average iron atom had five to six phosphorus neighbors at 3.17 A. Moreover, the number of detectable iron neighbors was lower when phosphate was high or present during reconstitution (2-3 vs 5-6), and the interatomic distance was longer (3.50 vs 3.03 A), indicating that some phosphate bridges neighboring iron atoms. However, the decrease in the number of detectable iron-iron neighbors compared to HSF and the higher number of Fe-P interactions relative to Fe-Fe interactions suggest that some phosphate ligands were chain termini, or blocked crystal growth, and/or introduced defects which contributed both to the long-range disorder and to altered redox properties previously observed in AV ferritin.