The hFbpABC Transporter from Haemophilus influenzae Functions as a Binding-Protein-Dependent ABC Transporter with High Specificity and Affinity for Ferric Iron

Abstract

ABSTRACT Pathogenic Haemophilus influenzae , Neisseria spp. ( Neisseria gonorrhoeae and N. meningitidis ), Serratia marcescens , and other gram-negative bacteria utilize a periplasm-to-cytosol FbpABC iron transporter. In this study, we investigated the H. influenzae FbpABC transporter in a siderophore-deficient Escherichia coli background to assess biochemical aspects of FbpABC transporter function. Using a radiolabeled Fe 3+ transport assay, we established an apparent K m = 0.9 μM and V max = 1.8 pmol/10 7 cells/min for FbpABC-mediated transport. Complementation experiments showed that hFbpABC is dependent on the FbpA binding protein for transport. The ATPase inhibitor sodium orthovanadate demonstrated dose-dependent inhibition of FbpABC transport, while the protonmotive-force-inhibitor carbonyl cyanide m -chlorophenyl hydrazone had no effect. Metal competition experiments demonstrated that the transporter has high specificity for Fe 3+ and selectivity for trivalent metals, including Ga 3+ and Al 3+ , over divalent metals. Metal sensitivity experiments showed that several divalent metals, including copper, nickel, and zinc, exhibited general toxicity towards E. coli . Significantly, gallium-induced toxicity was specific only to E. coli expressing FbpABC. A single-amino-acid mutation in the gene encoding the periplasmic binding protein, FbpA(Y196I), resulted in a greatly diminished iron binding affinity K d = 5.2 × 10 −4 M −1 , ∼14 orders of magnitude weaker than that of the wild-type protein. Surprisingly, the mutant transporter [FbpA(Y196I)BC] exhibited substantial transport activity, ∼35% of wild-type transport, with K m = 1.2 μM and V max = 0.5 pmol/10 7 cells/min. We conclude that the FbpABC complexes possess basic characteristics representative of the family of bacterial binding protein-dependent ABC transporters. However, the specificity and high-affinity binding characteristics suggest that the FbpABC transporters function as specialized transporters satisfying the strict chemical requirements of ferric iron (Fe 3+ ) binding and membrane transport.