Role of citrate and phosphate anions in the mechanism of iron(III) sequestration by ferric binding protein: kinetic studies of the formation of the holoprotein of wild-type FbpA and its engineered mutants.

Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrheae, Neisseria meningitidis, and Haemophilus influenzae. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with loading of iron(III) into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate, only two steps are discernible. A stable intermediate containing iron(III) citrate-bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggest...