JBIC Journal of Biological Inorganic Chemistry

, Volume 13, Issue 6, pp 887–898 | Cite as

Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA

  • Katherine D. Weaver
  • Jared J. Heymann
  • Arnav Mehta
  • Petra L. Roulhac
  • Damon S. Anderson
  • Andrew J. Nowalk
  • Pratima Adhikari
  • Timothy A. Mietzner
  • Michael C. Fitzgerald
  • Alvin L. Crumbliss
Original Paper


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 Fe3+ from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga3+, which, unlike Fe3+, is not redox-active. Because of a similar size and charge as Fe3+, Ga3+ is widely used as a non-redox-active Fe3+ substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga3+ and Fe3+, focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga3+ complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH2PO4 was determined by UV-difference spectroscopy as \( \log \,K_{{\text{eff}}}^\prime = 13.7 \pm 0.6. \) This represents a 105-fold weaker binding relative to Fe3+ at identical conditions. The unfolding/refolding behavior of Ga3+ and Fe3+ 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 Fe3+ and Ga3+ sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga3+FbpA–PO4 assembly, and the similarities/differences of stepwise loading of Fe3+ into apo- or Ga3+-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga3+ transport and toxicity in cell culture studies.


Binding constant Ferric binding protein Iron transport Gallium transport Kinetics 



A.L.C. thanks the NSF (CHE 0418006) for financial support. J.J.H. received partial support from an NIH CBTE training grant (T32GM8555).

Supplementary material

775_2008_376_MOESM1_ESM.pdf (255 kb)
Supplementary Material (PDF 254 kb)


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Copyright information

© SBIC 2008

Authors and Affiliations

  • Katherine D. Weaver
    • 1
  • Jared J. Heymann
    • 1
  • Arnav Mehta
    • 1
  • Petra L. Roulhac
    • 1
  • Damon S. Anderson
    • 2
    • 3
  • Andrew J. Nowalk
    • 3
  • Pratima Adhikari
    • 3
  • Timothy A. Mietzner
    • 3
  • Michael C. Fitzgerald
    • 1
  • Alvin L. Crumbliss
    • 1
  1. 1.Department of ChemistryDuke UniversityDurhamUSA
  2. 2.Molecular Cardiology Research InstituteTUFTS-New England Medical CenterBostonUSA
  3. 3.Department of Molecular Genetics and BiochemistryUniversity of Pittsburgh School of MedicinePittsburghUSA

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