The periplasmic FbpA (ferric-binding protein A) from Haemophilus influenzae plays a critical role in acquiring iron from host transferrin, shuttling iron from the outer-membrane receptor complex to the inner-membrane transport complex responsible for transporting iron into the cytoplasm. In the present study, we report on the properties of a series of site-directed mutants of two adjacent tyrosine residues involved in iron co-ordination, and demonstrate that, in contrast with mutation of equivalent residues in the N-lobe of human transferrin, the mutant FbpAs retain significant iron-binding affinity regardless of the nature of the replacement amino acid. The Y195A and Y196A FbpAs are not only capable of binding iron, but are proficient in mediating periplasm-to-cytoplasm iron transport in a reconstituted FbpABC pathway in a specialized Escherichia coli reporter strain. This indicates that their inability to mediate iron acquisition from transferrin is due to their inability to compete for iron with receptor-bound transferrin. Wild-type iron-loaded FbpA could be crystalized in a closed or open state depending upon the crystallization conditions. The synergistic phosphate anion was not present in the iron-loaded open form, suggesting that initial anchoring of iron was mediated by the adjacent tyrosine residues and that alternate pathways for iron and anion binding and release may be considered. Collectively, these results demonstrate that the presence of a twin-tyrosine motif common to many periplasmic iron-binding proteins is critical for initially capturing the ferric ion released by the outer-membrane receptor complex.

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