Specificity of the Synergistic Anion for Iron Binding by Ferric Binding Protein from Neisseria gonorrhoeae

Abstract

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.

Ferric ion (hydr)oxo clusters in the “Venus flytrap” cleft of FbpA: Mössbauer, calorimetric and mass spectrometric studies

“…10 5 M -1 (Table 2), which is close to the value reported previously for binding of a single iron to nFbpA using ITC [24]. The value of the apparent binding constant does not suggest very strong binding, but it is noted that the protein binds to Fe III from a Fe III -citrate complex, i.e.…”

“…7 times lower in our experiments, and the higher ratio of iron-to-protein FbpA:Fe (1:50) used in our studies compared to 1:25 in the literature report. Comparing our studies with those of Murphy et al [24] suggests that when other iron chelators such as citrate are lower in concentration and the iron(III) concentration is high, then the protein can bind multiple iron atoms in the interdomain binding cleft with an apparent binding constant 7.6 x 10 4 M -1 and H = -0.87 kcal mol 1 at pH 7.4 (vs. 1.6 x 10 4 M -1 , H = -0.46 kcal mol -1 , pH 7.0 in literature) [24]. Hence this suggests that although the protein has a higher affinity for iron compared to citrate, at sufficiently high citrate concentrations iron uptake by the protein can be inhibited.…”

“…8) showed good agreement with the experimental data. However it should be noted that the model used for reproducing the experimental spectra is a pure fit model and the parameters given in Studies of a single iron binding to nFbpA by ITC in the presence and absence of phosphate using 3.5 mM FeCl 3 and 30 mM citrate at pH 7.0 have been reported [24]. These gave a binding constant for Fe III -nFbpA in absence of phosphate of 1.6 x 10 4 M -1 .…”

Anion-independent Iron Coordination by the Campylobacter jejuni Ferric Binding Protein

“…In Class I FbpAs (hFbpA and nFbpA), phosphate and the two conserved Tyr residues form an iron half-site that is proposed to bind iron before forming a closed holo structure (32). Also, phosphate is necessary for the formation of the closed conformation (26). In mFbpA (Class II), three C-terminal Tyr residues form the analogous half-site, eliminating the need for an anion (phosphate) at this position (35); however, a carbonate anion is observed in the closed holo structure (Protein Data Bank code 1SI0) and is the only additional iron ligand (Fig.…”

“…All of the reagents used were purchased from Sigma-Aldrich. Holo nFbpA was obtained as described previously (26). All of the iron binding experiments were performed using 2 mg/ml (0.055 mM) apo protein.…”