Proton co-transport and voltage dependence enforce unidirectional metal transport in an Nramp transporter

Secondary transporters harness electrochemical energy to move substrate through structurally-enforced co-substrate \"coupling\". We untangle the \"proton-metal coupling\" behavior by a Natural resistance-associated macrophage protein (Nramp) transporter into two distinct phenomena: {Delta}pH stimulation of metal transport and metal stimulation of proton co-transport. Surprisingly, metal type dictates co-transport stoichiometry, leading to manganese-proton symport but cadmium uniport. Additionally, the membrane potential affects both the kinetics and thermodynamics of metal transport. A conserved salt-bridge network near the metal-binding site imparts voltage dependence and enables proton co-transport, properties that allow this Nramp transporter to maximize metal uptake and prevent deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp metal-proton symport in which, in addition to substrate gradients determining directionality as in canonical secondary transport, synergy between protein structure and physiological voltage enforces unidirectional substrate movement. Our results illustrate a functional advantage that arises from deviations from the traditional model of symport.