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

Abstract

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.