Abstract In all living organisms, transition metals are essential trace elements. Their unique chemical properties are exploited in metalloenzymes, metalloproteins, and as signaling ions, to perform key biochemical processes. Integrated metal uptake, distribution, storage, and sensing protein networks guarantee that essential metals are delivered to their correct targets, while toxic metals are selectively removed from the cellular environment. Transmembrane metal transporters and interacting metallochaperones are key hubs that selectively regulate the fluxes of metals across cellular membranes. The chemical, molecular and structural principles that govern metal transporter cargo selectivity, promiscuity, translocation, and substrate delivery by metallochaperones and associated metalloproteins are elusive. We developed a strategy that leverages integrated chemical, biophysical, and structural approaches to determine how metal selection and transport occurs in transporter families featuring different topologies, transport mechanisms, and energetics. This MIRA application targets the study of primary active metal pumps, secondary active solute carriers (SLC), and ion facilitators to address: i) the bioinorganic and coordination chemistry determinants controlling substrate selectivity, affinity, and kinetic lability for rapid translocation across membranes; ii) how conserved structural frameworks in transporter families are adapted to diversify cargo specificity towards essential 1st-row and toxic 2nd/3rd-row transition metals; iii) how energy transduction and conformational changes are coupled to substrate transport; and iv) the chemical, thermodynamic, kinetic, and structural principles that control metal delivery and uptake to/from transporters by metallochaperones. The program will target metal transporters involved in metal homeostasis, disease progression, and pathogen virulence: 1) copper and zinc P-type ATPase pumps and their metallochaperones, which control copper levels in humans and Cu/Zn concentrations in bacteria acting as virulence factors; 2) TMEM205, that we discovered as a new human copper/platinum transporter involved in Cu(I)-homeostasis and anti-cancer Pt-drug resistance, and TMEM52B as its putative chaperone; 3) Fe(II)-selective prokaryotic and eukaryotic transporters belonging to the IroT, ZIP and CDF families, responsible for iron acquisition and virulence in pathogens causing bacterial infections and neglected tropical diseases; and 4) prokaryotic nickel and cobalt transporters controlling essential acquisition of scarce trace elements for Ni/Co enzyme maturation. We will combine biophysical, spectroscopic, and structural transporter characterization in detergents with the study of substrate translocation events in real-time in native-like lipid bilayer systems, using the innovative fluorescence multi-probe platform that we established in our group. We expect to reveal new paradigms underlying transport kinetics, thermodynami...