We are seeking principles which govern biological activation of molecular oxygen. Our objective is a generalized understanding of mechanisms by which oxygen supports life in terms of structures of oxygenases, oxidases, oxygen, co-factors, and substrates. We propose to investigate selected dioxygenase, mixed function oxidases, and oxidase reactions in which copper, iron, (non-heme) and heme are prosthetic groups. We will continue our biochemical and biophysical studies by (1) determining the structures of active sites and related protein structures of copper, heme, and iron oxygenase and mixed function oxidases, (2) characterizing reactions which take place between these oxygenases, their substrates and O2, in terms of oxygen- and substrate-containing intermediates, (3) detecting and characterizing intermediates by specific transfer of (isotopic) oxygen to substrates, (5) establishing by kinetic and thermodynamic studies the sequence of formation and reaction of intermediates, and (6) separating the native subunits of beef heart cytochrome c oxidase, characterizing them chemically and physically, and reconstituting the functional aggregate oxidase with them both to establish the native character of the isolated subunits, and to determine the nature of the associations which occur when the functional enzyme is reconstituted from the subunits.
