Binding Protein A (FbpA) exists in the periplasmic space of various Gram-negative bacteria. Within these organisms, FbpA is responsible for iron sequestration and transport from the inside of the outer membrane to the outside of the cytosolic membrane. This iron sequestration and delivery parallels the function of mammalian transferrin (Tf), a genetic relative through the anion binding protein superfamily. The purpose of both FbpA and Tf is to deliver the essential nutrient iron, while inhibiting metal-catalyzed oxidative stress. Iron control by both FbpA and Tf is accomplished through thermodynamically stable binding using four amino acid side chain residues and an exogenous anion. While iron is considered the primary cargo for the periplasmic transporter FbpA, the exogenous or "synergistic" anion serves a significant role in determining FeFbpA-X thermodynamics. In this work, we examine the synergistic anion's effect on both the thermodynamics and kinetics of the FeFbpA-X complex.

We have first characterized the least stable known holo-FbpA complex, FeFbpA-SO₄, determining complex stability to be K'_eff = 16.2 and the FbpA-bound iron redox potential through spectroelectrochemistry to be -158 mV NHE. These values extend the range of known FeFbpA-X complexes in both stability and redox behavior to an approximate variation of 14 kJ mol^-1 as compared to the most stable, least easily reducible FbpA complex, FeFbpA-PO₄. Through the use of extra-thermodynamic relationships, we have determined that the modulation resulting from changes in synergistic anion identity may be caused by variations in FbpA///anion interactions, and properties of the anion such as electrostatics and hydration enthalpy. These results are further supported by an analysis of anion binding to apo-FbpA and a comparison of this anion binding activity to the stability of the overall complex. These results are further extended to understand the thermodynamics and kinetics of binding and controlling iron from low molecular weight sources.

The ability of the anion to modulate FeFbpA-X thermodynamics in vitro may be significant in understanding the Gram-negative bacterial iron uptake system, but in order for the anion to hold such prominence, it is necessary for FbpA to permit kinetically labile anion exchange. Here, anion exchange kinetics are discussed, examining not only the mechanism for numerous anions, but also the significance of the anion binding site, the role of background environmental anion identity in the context of the Hofmeister series, and the possible importance of the active site non-specific anion binding residues. Our results show that the synergistic anion within FeFbpA-X is kinetically labile, and capable of exchanging for a variety of anions, while maintaining a thermodynamically stable FeFbpA-X complex. We have classified the behavior of anion exchange to be of two groups, Groups 1 and 2, defined by the properties of the entering anion, particularly the ability of this anion to independently chelate iron. In all cases, however, starting with FeFbpA-SO₄ and FeFbpA-NTA, anion exchange was observed as the first step of the reaction.

While iron is the natural substrate of FbpA, chemically similar metals such as the gallium 3+ ion may also bind to this protein. Through competition techniques, we have determined the GaFbpA-PO₄ stability to be five orders of magnitude lower in stability than the comparable iron complex, and have shown the bound gallium to be kinetically exchangeable for iron, with a similar mechanism of iron loading to apo-FbpA. Through in vitro and in vivo experimentation, we will also explore the origins of substrate selectivity of the protein.

Overall, this work focuses on the contribution of individual interactions within the ternary complex components; Fe///X, Fe///FbpA, and FbpA///X, with an emphasis on the properties of these interactions responsible for the kinetics and thermodynamics of FeFbpA-X activity, and ultimately, biological function.