Function and assembly of heme-copper oxidases in aerobic respiratory chains

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All cells engage in a process of energy conversion in which food is broken down and some energy is harvested. This energy is then used to fuel biochemical synthesis, nutrient transport and cellular mobility. There are a set of enzymes that act to catalyze the processes of energy transfer and the study of these enzymes and their mechanisms of action make up a large part of the field of bioenergetics. For organisms that use oxygen for respiration a large fraction of energy conversion derives from the process of the conversion of oxygen to water. This reaction is catalyzed in aerobic species by a member of the heme-copper oxidase family of enzymes. Members of the heme-copper oxidase family are distinguished by an oxygen reaction site that includes a heme moiety and a copper ion that are closely spaced and highly cooperative n their reaction with oxygen. The research in this proposal is aimed at understanding the molecular mechanism used by the heme-copper oxidase family of enzyme to convert oxygen to water, and the harvesting of energy from this process. We use a combination of spectroscopic and kinetic approaches along with recombinant DNA methods to make mutant enzymes to unravel the physical details of these mechanisms to atomic detail. A second aspect of this proposal is to understand how these catalysts, which are essentially molecular machines, are put together. The process of assembly for heme-copper oxidases is intrinsically complex as it involves the assembly of several protein subunits and the simultaneous insertion of four reactive metal centers. The regulation of this process is important for its success. We are interested particularly in the delivery and incorporation of copper ions into the heme-copper oxidase complex. This process involves a specific copper binding protein that acts to chaperone the metal ion into its site of action in the heme-copper enzyme. Our studies will give insight into the functional aspects of energy transfer and how these systems are assembled. A central aspect of our progam is training highly qualified personnel in the application of biophysical approaches to understaniding biochemical mechanisms and to place their findings into the context of a biological system.