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

Abstract

Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenza. 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 iron(III) loading 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 suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron-loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics, and reduce iron sequestration ability. H9E, E57D, and E57Q have near native iron sequestration behavior; however iron binding rates are altered enabling assignment of sequential side chain interactions. Additionally, this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as “naked” or free iron as originally proposed.

Iron transport across the blood–brain barrier: development, neurovascular regulation and cerebral amyloid angiopathy

“…A single ferric iron atom can reversibly bind either lobe of Tf (C- or N-lobe) with high affinity ( ∼ 10 22 M -1 at pH 7.4) [16]. Release of iron from Tf can be potentiated through decreased pH, exogenous ligands, and the binding of Tf to its receptor [17-19] (see “Transferrin and transferrin receptor recycling-dependent BMVEC iron uptake pathways” section for details). In the brain, both Tf transcript and protein have been identified (Table 1).…”

“…Recently, our lab demonstrated that the reduction of iron from Tf, as measured by the colorimetric ferrozine assay [86], requires the binding of holo-Tf to the TfR on the cell surface of hBMVEC [40]. In addition to the contributions of TfR, an exogenous ligand will increase the E ° of the Tf-bound iron and thus increase the potential for reduction [18, 19]. The presence of the exogenous ligand citrate is physiologically relevant to serum [81, 87-89] and ISF [90].…”

Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity

“…12,25 This is quite distinct from the synergistic anion role that citrate can play for the ferric binding protein A (FbpA), which has a metal binding site similar to sTf. 24 …”

A ubiquitous metal, difficult to track: towards an understanding of the regulation of titanium(iv) in humans

“…This is a significant finding because others have argued that a metal-bound closed protein conformation is a requirement for metal delivery into cells by sTf. 39 Also significant is the synergistic role that citrate potentially plays in sTf transport of Ti(IV) because while metal transport is a function that it exhibits in bacteria, 45, 46 it has been little explored in humans. Further insight into the contribution of citrate and sTf to the transport and potential function of Ti(IV) is obtained by examining the efforts to produce a Ti(IV)-based anticancer therapeutic.…”