Project Summary/Abstract: This equipment supplement project proposal seeks funds to purchase a Cold-Spray Ionization Mass Spectrometer to carry out CSI-MS experiments, a powerful approach to characterizing typically unstable reactive intermediates. The scientific objectives include the design, synthesis & investigation of synthetic models which will aid the elucidation of fundamental aspects of structure, M-ligation, spectroscopy and reactivity relevant to copper and heme/M (M = Cu, Fe) processing of molecular oxygen (O2(g)) and nitric oxide (NO(g)). Copper proteins of concern include lytic polysaccharide monooxygenases, Cu-methane monooxygenases, the enzyme family which includes peptidylglycine monooxygenase, and a binuclear copper protein, NspF. Biochemical research has raised questions concerning the nature of their active sites and the mechanism(s) of action involving O2(g) activation and C-H hydroxylation. LPMOs may be peroxygenases, new pMMO studies suggest a mono-Cu active site, and it is now questioned as to whether DBM and PHM activate O2 with a Cu vs a Cu2 center. There are clear needs to synthesize and characterize the copper(II)-oxyl (CuII-O·) species; it has the oxidizing ability needed for the difficult biological substrates. We also plan to elucidate fundamentals critical to the O-O reductive cleavage process occurring in proteins which process O2. Also, we will generate and characterize the structures, physical properties and reactivity of new high-valent binuclear Cu(II)-O-Cu(III) complexes. Proposed research will also focus on the heme-copper active site of cytochrome c oxidases, where O2-binds and is reductively cleaved to give two mole-equiv water. The study of synthetic models aids an understanding of structure, O2-binding, proton or H-bonding facilitated O-O cleavage, and the role of the active-site phenol. Investigations are proposed to further investigate the mechanisms of O-O cleavage in heme-peroxo-copper constructs, where the porphyrinate, the Fe axial ligand and the copper ligand are systematically varied. A variety of planned approaches include study of new chelates for copper which possess three N-donors and an appended phenol. NO(g) synthetic model chemistry sub-projects with copper and heme-M will also be carried out. With Cu complexes, the focus will be on NO(g) reductive coupling, and study of mechanisms pertaining to the NO(g) metal-binding, formation of the N–N bond giving putative hyponitrite N2O22– intermediates, and proton and/or H-bonding contributions to N–O cleavage and N2O formation. Heme/Fe (or Cu) mediated NO(g) coupling is critical in NO-Reductases and synthetic models for this process will be investigated. Metal-peroxynitrite (PN, from metal ion + O2(g) + NO(g)) reactivity, especially toward CO2, will also be studied as relevant to biological activity.