Transport of Degraded Ferric Enterobactin by Fiu

Under iron deficient conditions, Gram‐negative bacteria like Escherichia coli secrete enterobactin, a catecholate siderophore that complexes and solubilizes iron in the extracellular environment. Ferric enterobactin (FeEnt) is the most avid natural or synthetic iron complex. It may be glucosylated (FeGEnt), and it is also subject to both chemical degradation and oxidation (FeEnt*). Neverthless, bacterial outer membrane transporters acquire FeEnt, FeGEnt and FeEnt*. For example, the E. coli Fiu protein is known to catalyze the uptake of monomeric catecholate iron complexes, and using siderophore nutrition tests and radioisotopic iron accumulation experiments, we found that Fiu preferentially recognizes and transports FeEnt*. To further analyze the specificity of the FeEnt* uptake process, we created Cys substitutions in the external surface loops of Fiu and labeled them with fluorescein maleimide (FM). During binding of ferric siderophores, the surface loops undergo conformational motion that quenches attached fluorophores, allowing quantitative determinations of affinity and specificity. We measured the affinity of Fiu‐FM for different ferric catecholate compounds: FeEnt*, FeEnt, and FeGEnt produced K d values of 0.46, 20, and 0.5 μM, respectively. Thus, Fiu manifests specificity for the degradation products of FeEnt, but also mediates the uptake of FeEnt and FeGEnt, identifying it as an important component of bacterial iron acquisition systems that are essential to bacterial survival. Support or Funding Information National Institutes of Health