Proposed investigations derive from two on-going R01 GM research programs. The study 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 (LPMOs), particulate methane monooxygenases (pMMOs), the enzyme family which includes dopamine beta-monooxygenase (DBM) and peptidylglycine monooxygenase (PHM), and a coupled binuclear copper protein, NspF. Recent 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 (H2O2 co-substrate), 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 versus a Cu2 center process. Relevant to these issues, there are clear needs to synthesize and characterize the thus-far elusive copper(II)-oxyl (CuII-O) species; it has the oxidizing ability needed for the difficult LPMO or pMMO substrates. We also plan to elucidate fundamentals critical to the O-O reductive cleavage process occurring in all monooxygenases (& oxidases). Also, we will generate and characterize the structures, physical properties and reactivity of new high-valent binuclear Cu(II)-O-Cu(III) or related complexes. Otherwise, proposed research will focus on the heme-copper active site present in cytochrome c oxidases, where O2-binds and is reductively cleaved to give two mole-equiv water. The study of synthetic models can help elucidate details and aid an understanding of structure, O2-binding, proton or H-bonding facilitated O-O cleavage, and the role of the active-site phenol (in the His-Tyr cofactor) as a proton-electron donor. 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 especially the ligand for copper ion, are systematically varied. A variety of approaches are planned, including 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 copper complexes, the focus will be on NO(g) reductive coupling, and investigation of mechanisms pertaining to the NO(g) binding to metal ion(s), formation of the N?N bond giving putative hyponitrite N2O22? intermediates, and proton and/or H-bonding contributions to N?O cleavage and formation of N2O. Heme/Fe (or Cu) mediated NO(g) reductive coupling is critical in NO-Reductases and chemistry 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 in relation to the relevant biological activity.
The proposed investigations address fundamental aspects of the chemistry of copper and heme-copper coordination complexes with molecular oxygen and nitrogen oxides such as nitric oxide. The interactions of these biologically important gaseous small molecules, especially at metalloenzyme active-sites, are critical in normal functioning and health. Potential long-term applications of this basic research include development of enzyme inhibitors as drugs and relevant disease therapeutic strategies.
