Kinetics and Mechanism of Iron(III) Complexation by Ferric Binding Protein: The Role of Phosphate

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Abstract
Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp). This requires Fe3+ loading of Fbp at the inner leaflet of the outer membrane. A synergistic anion is required for tight Fe3+ sequestration by Fbp. Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro. Here, we report the kinetics and mechanism of Fe3+ loading of Fbp from Fe(NTA)aq in the presence of phosphate at pH 6.5. The reaction proceeds in four kinetically distinguishable steps to produce Fe3+Fbp(PO4) as a final product. The first three steps exhibit half-lives ranging from ca. 20 ms to 0.5 min, depending on the concentrations, and produce Fe3+Fbp(NTA) as an intermediate product of significant stability. The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate. Conversion of Fe3+Fbp(NTA) to Fe3+Fbp(PO4) in the fourth step is a slow process (half-life ∼ 2 h) and is facilitated by free phosphate. A mechanism for the Fe3+-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles. These data suggest that not only is a synergistic anion required for tight Fe3+ sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe3+ into the Fbp binding site.