FE2+ AND PHOSPHATE INTERACTIONS IN BACTERIAL FERRITIN FROM AZOTOBACTER-VINELANDII

Fe2+ binding to both apo- and holo- bacterial ferritin from Azotobacter vinelandii (AVBF) was measured as a function of pH under carefully controlled anaerobic conditions. Fe2+ binding to apo-AVBF is strongly pH dependent with 25 Fe2+ ions/apo-AVBF binding tightly at pH 5.5 and over 150 Fe2+/apo-AVBF at pH 9.0. Holo-AVBF gave a similar pH-dependent binding profile with over 400 Fe2+/AVBF binding at pH of 9.0. Proton release per Fe2+ bound to either AVBF protein increases with increasing pH until a total of about two protons are released at pH 9.0. These binding results are both qualitatively and quantitatively different from corresponding measurements (Jacobs et al., 1989) on apo- and holo- mammalian ferritin (MF) where less Fe2+ binds in both cases. The high level of Fe2+ binding to holo-AVBF relative to that of mammalian ferritin is a consequence of the higher phosphate content in the core of AVBF. Reduction of AVBF by either dithionite or methyl viologen in the absence of chelating agents demonstrated that phosphate, but not Fe2+, is released from the AVBF core in amounts comensurate with the degree of iron reduction, although even at 100% reduction considerable phosphate remains associated with the reduced mineral core. Fe2+ binding to holo-AVBF made deficient in phosphate was lower than that of native AVBF, while the addition of phosphate to native holo-AVBF increased the Fe2+ binding capacity. These results clearly support the role of phosphate as the site of interaction of Fe2+ with the AVBF mineral core. Mossbauer measurements of Fe-57(2+) bound to holo-AVBF under controlled anaerobic conditions indicate that the bound Fe2+ undergoes a reversible electron-transfer reaction with the mineral core iron and forms Fe-57(3+), presumably bound to the mineral core surface. The phosphate present on the mineral core is involved in catalyzing this internal electron-transfer reaction, a process which may be an important step in bacterial ferritin mineral core development.