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

• Published in: Journal of biological inorganic chemistry 2008-08, Vol.13 (6), p.887-898
• Main Authors: Weaver, Katherine D., Heymann, Jared J., Mehta, Arnav, Roulhac, Petra L., Anderson, Damon S., Nowalk, Andrew J., Adhikari, Pratima, Mietzner, Timothy A., Fitzgerald, Michael C., Crumbliss, Alvin L.
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Ferric Compounds - chemistry; Ferric Compounds - metabolism; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Fluorescent Dyes - pharmacology; Gallium - chemistry; Gallium - metabolism; Gallium - pharmacology; Haemophilus influenzae - drug effects; Haemophilus influenzae - growth & development; Iron-Binding Proteins - chemistry; Iron-Binding Proteins - isolation & purification; Iron-Binding Proteins - metabolism; Kinetics; Life Sciences; Microbial Sensitivity Tests; Microbiology; Original Paper; Periplasmic Binding Proteins - chemistry; Periplasmic Binding Proteins - isolation & purification; Periplasmic Binding Proteins - metabolism; Sensitivity and Specificity; Spectrophotometry, Ultraviolet - methods; Thermodynamics
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-008-0376-5

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 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.