The different affinity of the two metal-binding sites of human ferroportin drives outward directionality of transport.

Ferroportin, the only known cellular iron exporter, belongs to the major facilitator superfamily of transporters, which cycle between inward-open, occluded and outward-open conformations to translocate substrates across membranes. Recently reported cryoEM structures of ferroportin identified two metal-binding sites in the central cavity of the protein, with site S1 that includes residues D39 and H43, while site S2 is formed by C326 and H507. Here we have employed fluorescence spectroscopy to evaluate the binding affinity for cobalt of human ferroportin. The results suggest that S2 has a higher affinity for cobalt than S1. Results are discussed in view of available structural data on the outward-open conformation of Fpn and of a novel structural model of the inward-open conformation, obtained with a custom implementation of AlphaFold 2. We propose a mechanism by which the outward flux of iron could be driven by the different affinity of the two sites.