Cytochrome ba3 from Thermus thernzophilus, with a sequence identity to other enzymes of less than 20 percent, is among the most divergent of the heme-copper oxidases, yet it performs the core functions of the heme-copper oxidase super family. It is thus a storehouse of natural mutations that endow the protein with novel chemical behaviors and can be used to evaluate energy transduction mechanisms. Two major accomplishments in our laboratory support a new direction for the research: (a) Development of an E. coli-based expression system for Thermus cytochrome C552, the natural substrate of ba3, that yields recombinant protein having structural and functional properties identical to those of native C552. (b) Development of a shuttle vector that over expresses cytochrome ba3 -4 fold in Thermus cells and is useful for the manipulation of cytochrome ba3 genes in E. coli. This 'system' will permit us to selectively alter Thermus DNA sequences using standard mutagenesis techniques, then to express the mutant proteins in their natural environment. Because we also know the structures of cytochrome C552 and of cytochrome ba3, these new tools permit us to begin studies of enzyme mechanism. Four Specific Aims are suggested: (1) We will examine the mechanism of ba3 under near-physiological conditions to include recording of transient optical absorption and MCD spectral changes that occur during oxidation of reduced ba3 by O2, transient resonance Raman spectroscopy to identify Fea3-oxygen intermediates, and correlation with the time course of proton uptake. (2)We will use EXAFS and FTIR to examine potential changes in coordination at CuB in CuA deficient- ba3 and explore possible chloride binding to CUB. The results of these studies will define the number and chemical nature of intermediates and the times of their appearance and disappearance during reoxidation. The X-ray absorption studies will provide fundamental new knowledge about coordination changes at CUB in enzyme function.(3)We propose to initiate a comparative crystallographic effort with Dr. Duncan McRee at the Scripps Research institute with the goal of obtaining structures of fully reduced and carbonylated forms of cytochrome ba3, the mutant CuA def -protein and a complex of oxidized cytochromes C552 and ba3. (4)We propose the technical goal of refining our ba3 -expression system to include the capability of easily preparing site-selective mutations in both subunits II and I of cytochrome ba3. The significance of this work to human health lies in providing fundamental new information about conservation of energy by biological systems.