PROJECT SUMMARY Copper is an essential trace element utilized by eukaryotic cells as an enzymatic redox cofactor. At high concentrations the metal is toxic, and complex homeostatic mechanisms have developed to ensure that it is maintained at the correct physiological level. Prokaryotes such as gram negative pathogens have a diminished requirement for the metal, and show sensitivity to copper at levels well tolerated by mammalian cells. For this reason, macrophages utilize pathogenic copper toxicity as part of their defense against invaders while the latter combat the host-defense by upregulating their exporter machinery. Our research is focused on the mechanisms of copper transport and catalysis and is divided into three separate but overlapping projects. Project 1 examines the reaction mechanism of mononuclear monooxygenases exemplified by peptidylglycine monooxygenase (PHM), the only enzyme capable of installing the critical post-translational amide into peptide hormones. We have identified three important unanswered questions – (i) the pathway for electron transfer (ii) the chemical speciation of reaction intermediates and (iii) the mechanism of substrate triggering, and have designed an approach for interrogating each. Project 2 focuses on the mechanism of pathogenic copper export via a study of the CusCBAF exporter of E. coli. Here we use a novel labeling approach to track the flux of metal ions as they move through the pump. We build on recent results that have successfully determined the role CusF and CusB in metalating CusA, the rates of copper transfer from CusF to CusB, and the identity of the CusF-CusB complex as a shared ligand intermediate. Unanswered questions include the mechanism of CusF to CusA metal exchange, the role of metal-binding residues in selectivity and efficiency, and the function of the novel His2Phe Cu(I)/Ag(I) binding site in the CusS regulator. Project 3 takes our program into new territory by investigating structure/function of the newly discovered copper- dependent kinases MEK1/2 and ULK1/2 involved in cell signaling and tumorogenesis. The integrated approach to all three projects leverages the Pi's track record in developing toolsets that can interrogate the chemistry of copper, but particularly the Cu(I) state which reacts with oxygen, and is the form in which copper is trafficked in the cell. Two aspects of this effort are of particular utility: (i) development of metal-directed probes of Cu(I) coordinate structure utilizing x-ray absorption and emission spectroscopies and (ii) development of labeling strategies utilizing selenomethionine substitution coupled to Se K EXAFS. The expected outcome of the research is a better understanding of how biology leverages the fundamental chemistry of copper to achieve functionality, recognizing that function requires balance between the requirements for selective metalation, the structural determinants of catalysis, and pathogenic virulence induced by robust bacterial exp...