Cytochrome c oxidase is an enzyme that provides energy for the cell. It does so by coupling dioxygen reduction to proton pumping leading to oxidative phosphorylation. The cytochrome ba3 from Thermus thermophilus is a distant homolog of the mammalian enzymes that contains a storehouse of natural mutations that, while retaining an almost identical three-dimesional structure, confer on the enzyme novel chemical behaviours that provide unique insight on function. During past funding periods we developed a homologous expression system, learned how to make mutant forms, demonstrated the presence of a single pathway for access of protons into the active site, developed efficient methods to obtain X-ray diffracting crystals, determined the structures of several novel forms of the enzyme, introduced two very powerful forms of spectroscopy to examine the enzyme in single crystals, and published, as hypothesis, a detailed chemical mechanism for coupling proton pumping with dioxygen reduction. Here we propose four Specific Aims: [1] to explore the mechanism of the enzyme using rapid measurement of O- isotope shifted resonance Raman spectra. [2] Combine structure-directed, site-directed mutation to regulate the several structural pathways whereby substrates (electrons, dioxygen, water and protons) enter and leave the active site. Our hope to trap new intermediates for detailed chemical study. In Aims [3&4] we focus on obtaining the structures of oxygen-intermediates that occur in the overall mechanism. The significance of this work to human health lies in providing fundamental information about the conservation of energy in biological systems. PUBLIC HEALTH RELEVANCE: Cytochrome c oxidase is an enzyme that helps power the eukaryotic cell. It combines electrons from the food we eat, oxygen from the air we breath, and protons from the fluid of which we are composed and creates an extreme proton gradient across the mitochondrial membrane. The most common use of this gradient is its capture and dissipation by the ATP synthase that provides the ATP to drive our metabolism. The chemistry whereby cytochrome c oxidase generates the proton gradient remains one of the deepest and most mysterious of biological chemistry. The purpose of our research is to understand this mechanism in respiration at the molecular level.