title: Sulfate as a synergistic anion facilitating iron binding by the bacterial transferrin FbpA: the origins and effects of anion promiscuity.
abstract: The ferric binding protein, FbpA, has been demonstrated to facilitate the transport of naked Fe3+ 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 Fe3+ by FbpA in the presence of sulfate, at an assumed periplasmic pH of 6.5 to form FeFbpA-SO4 with K'(eff) = 1.7 x 10(16) M(-1) (at 20 degrees C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO4 protein assembly is 2 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-SO4 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 Fe3+ 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-SO4 with phosphate and arsenate, where first-order kinetics with respect to FeFbpA-SO4 (k = 30 s(-1)) are observed 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.
authors.affiliation: Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Heymann
authors.forename: K D
authors.initials: KD
authors.lastname: Weaver
authors.forename: T A
authors.initials: TA
authors.lastname: Mietzner
authors.forename: A L
authors.initials: AL
authors.lastname: Crumbliss
mesh_terms: D000838:Anions / Q000737:chemistry; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D004563:Electrochemistry; D007501:Iron / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009344:Neisseria gonorrhoeae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013431:Sulfates / Q000737:chemistry*; D013816:Thermodynamics; D014168:Transferrin / Q000737:chemistry*
chemical_list: D000838:Anions; D001059:Apoproteins; D008024:Ligands; D013431:Sulfates; D014168:Transferrin; D007501:Iron