The ferric binding protein, FbpA, has been demonstrated to facilitate the transport of naked Fe 3+ across the periplasmic space of several gram-negative bacteria. The sequestration of iron by FbpA is facilitated by the presence of a synergistic anion, such as phosphate or sulfate. Here we report the sequestration of Fe 3+ by FbpA in the presence of sulfate, at an assumed periplasmic pH of 6.5 to form FeFbpA-SO 4 with K′ eff = 1.7 × 10 16 M −1 (at 20°C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO 4 protein assembly is two orders of magnitude lower than when bound with phosphate and is the lowest of any of the FeFbpA-X assemblies yet reported. Iron reduction at the cytosolic membrane receptor may be an essential aspect of the periplasmic iron transport process and with an E 1/2 of −158 mV (NHE), FeFbpA-SO 4 is the most easily reduced of all FeFbpA-X assemblies yet studied. The variation of FeFbpA-X assembly stability (K′ eff ) and ease of reduction (E 1/2 ) with differing synergistic anions X n− are correlated over a range of 14 kJ, suggesting that the variations in redox potentials are due to stabilization of Fe 3+ in FeFbpA-X by X n− . Anion promiscuity of FbpA in the diverse composition of the periplasmic space is illustrated by the ex vivo exchange kinetics of FeFbpA-SO 4 with phosphate and arsenate, with first order kinetics with respect to FeFbpA-SO 4 (k = 30 s −1 ) at pH 6.5, independent of entering anion concentration and identity. Anion lability and influence on the iron affinity and reduction potential for FeFbpA-X support the hypothesis that synergistic anion exchange may be an important regulator in iron delivery to the cytosol. This structural and thermodynamic analysis of anion binding in FeFbpA-X provides additional insight into anion promiscuity and importance.

The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.

A group of five graduate students and a faculty mentor used the cultural popularity of forensics to develop a first-year undergraduate seminar. This course fulfilled two main objectives: First, the graduate student instructors developed professionally through a two-year process of creating, instructing, and revising a course. Second, a variety of pedagogical techniques including lectures, in-class demonstrations, laboratories, student presentations, and mock criminal investigations were used to teach an introductory-level forensics seminar course with substantial chemistry content. Herein, we share our experiences in developing an effective course structure in which students were introduced to science through forensics, with an emphasis on the development of essential skills, including critical thinking and public speaking. An assessment of the project's impact on graduate students, undergraduate students, and the department is also provided.

The bacterial transferrin ferric binding protein A (FbpA) requires an exogenous anion to facilitate iron sequestration, and subsequently to shuttle the metal across the periplasm to the cytoplasmic membrane. In the diverse conditions of the periplasm, numerous anions are known to be present. Prior in vitro experiments have demonstrated the ability of multiple anions to fulfill the synergistic iron-binding requirement, and the identity of the bound anion has been shown to modulate important physicochemical properties of iron-bound FbpA (FeFbpA). Here we address the kinetics and mechanism of anion exchange for the FeFbpA-nitrilotriacetate (NTA) assembly with several biologically relevant anions (citrate, oxalate, phosphate, and pyrophosphate), with nonphysiologic NTA serving as a representative synergistic anion/chelator. The kinetic data are consistent with an anion-exchange process that occurs in multiple steps, dependent on the identity of both the entering anion and the leaving anion. The exchange mechanism may proceed either as a direct substitution or through an intermediate FeFbpA-X* assembly based on anion (X) identity. Our kinetic results further develop an understanding of exogenous anion lability in the periplasm, as well as address the final step of the iron-free FbpA (apo-FbpA)/Fe(3+) sequestration mechanism. Our results highlight the kinetic significance of the FbpA anion binding site, demonstrating a correlation between apo-FbpA/anion affinity and the FeFbpA rate of anion exchange, further supporting the requirement of an exogenous anion to complete tight sequestration of iron by FbpA, and developing a mechanism for anion exchange within FeFbpA that is dependent on the identity of both the entering anion and the leaving anion.