title: Kinetics and mechanism of iron release from the bacterial ferric binding protein nFbp: exogenous anion influence and comparison with mammalian transferrin.
abstract: Ferric binding protein, Fbp, serves an essential biological function in shuttling naked (hydrated) Fe(3+) across the periplasmic space of many Gram-negative bacteria. In this process, iron must be released at the cytoplasmic membrane to a permease. How iron is released from Fbp has yet to be resolved. Consequently, understanding the dynamics of iron release from Fbp is of both biological and chemical interest. Fbp requires an exogenous anion, e.g. phosphate when isolated from cell lysates, for tight iron sequestration. To address the role of exogenous anion identity and lability on Fe(aq)(3+) dissociation from Fbp, the kinetics of PO(4)(3-) exchange in Fe(3+) nFbp(PO(4)) ( nFbp=recombinant Fbp from Neisseria meningitidis) were investigated by dynamic (31)P NMR and the kinetics of Fe(3+) dissociation from Fe(3+) nFbp(X) (X=PO(4)(3-), citrate anion) were investigated by stopped-flow pH-jump measurements. We justify the use of non-physiological low-pH conditions because a high [H(+)] will drive the Fe(aq)(3+) dissociation reaction to completion without using competing chelators, whose presence may complicate or influence the dissociation mechanism. For perspective, these studies of nFbp (which has been referred to as a bacterial transferrin) are compared to new and previously published kinetic and thermodynamic data for mammalian transferrin. Significantly, we address the lability of the Fe(3+) coordination shell in nFbp, Fe(3+) nFbp(X) (X=PO(4)(3-), citrate), with respect to exogenous anion (X(n-)) exchange and dissociation, and ultimately complete dissociation of the protein to yield naked (hydrated) Fe(aq)(3+). These findings are a first step in understanding the process of iron donation to the bacterial permease for transport across the cytoplasmic membrane.
authors.affiliation: Department of Chemistry, Duke University, Durham, NC 27708-0346, USA.
authors.forename: Hakim
authors.initials: H
authors.lastname: Boukhalfa
authors.forename: Damon S
authors.initials: DS
authors.lastname: Anderson
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Mietzner
authors.forename: Alvin L
authors.initials: AL
authors.lastname: Crumbliss
mesh_terms: D000838:Anions; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D007501:Iron / Q000493:pharmacokinetics*; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D009345:Neisseria meningitidis; D014168:Transferrin / Q000378:metabolism*
chemical_list: D000838:Anions; D027682:Cation Transport Proteins; D033862:Iron-Binding Proteins; D014168:Transferrin; D007501:Iron