Dysregulation of copper homeostasis has multiple consequences for human health. Mammalian cells use copper pumps to maintain homeostasis, metalate copper-dependent enzymes, and transfer copper from one organelle/cell/tissue to another. Of equal significance is the study of bacterial transporters and their role as virulence factors in pathogens. An important class of efflux pump, present in a number of gram-negative pathogenic organisms is the RND- type heavy metal exporter exemplified by the CusCBAF system of E. coli which spans the periplasmic space and exhibits selectivity for export of Cu(I) and Ag(I). The complex is comprised of three proteins, CusA a transmembrane pump, CusB is a soluble “adaptor” protein, and CusC, an outer-membrane pore. In addition, the export machinery requires the presence of CusF, a small soluble periplasmic chaperone which shuttles Cu or Ag to the tripartite complex. Despite many advances in recent years, a critical piece of the puzzle has been missing – the rate at which metals transfer, and how that rate is controlled by specific structural elements of the interacting protein components. The PI’s laboratory has developed a suite of kinetic tools involving selenomethionine (SeM) substitution coupled to rapid freeze quench (RFQ) mixing and XAS detection which are broadly applicable. This proposal sets out to apply these tools to study the mechanisms of metal export by the CusCBAF system in molecular detail. There are three specific aims. Aim 1 will explore the unique coordination chemistry of CusF, which includes an unprecedented tryptophan ligand (W44) that caps the site and gives rise to fluorescence emission at 490 nm. The novel finding that CO binds to the W44A variant will be used to test the hypothesis that the role of W44 is to protect the site from oxidation via O2. Aim 2 will use RFQ mixing coupled to XAS detection of Se-Cu/Ag at the Se edge to follow the kinetics of metal transfer from CusF to CusB and from CusF to CusA with the goal of determining residues important for the efficiency of metal export. Transfer complexes predicted from computational studies of protein-protein interactions will be isolated and characterized. Aim 3 will investigate the mechanism of metal sensing by the periplasmic domain of the histidine kinase CusS which activates the CusR transcription factor via autophosphorylation and phosphotransfer, testing the hypothesis that the sensory domain uses high and low-affinity metal binding sites to switch on and upregulate its kinase activity. The expected outcome is an understanding of the mechanism of metal export in molecular detail, a prerequisite for the development of strategies for interrupting pathogenic metal resistance pathways, and diminishing virulence.