Abstract Transition metals are essential trace elements for life, playing pivotal roles in biochemical processes. First-row transition metals, such as iron, copper, and zinc, play fundamental catalytic, structural and signaling functions. Their concentrations are tightly regulated to meet indispensable cellular requirements without reaching toxic levels. On the other hand, non-essential second-/third-row transition metal complexes are toxic to cells and exploited as therapeutic molecules. A gatekeeper role in controlling metal concentrations in all cells is mediated by transmembrane transporters that regulate the vectorial metal uptake and extrusion across cellular membranes. The principles underlying metal selectivity and molecular mechanism of transport by these nanomachines remain elusive. This MIRA application targets the study of primary active transition metal pumps and solute carriers (SLC) and will focus on: (i) investigating the principles of metal selectivity for first, second- and third- row transition metals and (ii) their metal coordination chemistry; (iii) determining the metal translocation pathway; (iv) addressing the mechanisms of energy transduction processes at a molecular level. My laboratory has developed an integrated chemical, biophysical, and structural approach to determine how metal selection and transport occurs in different metal transporter families. With this multidisciplinary strategy, we will target known and novel transporter families involved in metal homeostasis and in disease progression; specifically: 1) P1B-type ATPases, primary active transporters controlling intracellular copper levels in humans, and modulating the concentrations of copper and other transition metals in pathogenic bacteria; 2) TMEM205, a novel human transporter potentially involved in copper extrusion and responsible for anti-cancer Pt-complexes transport and resistance; 3) IroT transporters, putative iron-regulated solute carriers responsible for iron(II) acquisition and virulence in pathogenic prokaryotes. We will couple biophysical, spectroscopic, and structural studies on purified, detergent-solubilized transporters to the characterization in proteoliposomes, where the transporters are embedded in a native-like lipid bilayer. By encapsulating in the proteoliposomes metal-dependent fluorescence chelators, sensors for secondary ions, and probes for membrane potential, we will develop an in vitro tool for monitoring real-time substrate translocation in a membrane environment. This platform establishes an innovative framework to address i) the metal substrate selectivity, ii) the nature of cotransported ions, iii) their relative stoichiometry, iv) the electrogenic properties, and v) the role of membrane potential on catalytic metal translocation. The project targets a neglected aspect of bioinorganic chemistry towards the understanding of the principles controlling metal translocation across membranes. Besides shading light on the basic molecular mechanisms governing metal transport, the study of novel targets will have a major impact on translational discoveries.

Public health relevance statement Transition metal translocation across cellular membranes mediated by transmembrane transporter proteins is a central process regulating intracellular essential and toxic metal concentrations within cells. The project will establish a platform to reveal, at a molecular level, the principles controlling transition metal selectivity and translocation in different metal ion pumps and solute carrier protein families that are central to cellular physiology, disease, and therapy.