Most aerobic life on earth depends upon the functions of the enzyme superfamily known as the heme-copper oxidases, which comprise the terminal oxidase systems of cellular respiration in all plants and animals and many simpler organisms. The central aim of this proposal is to apply static infrared spectroscopy and time-resolved infrared (TRIR) techniques (along with the complementary vibrational Raman approaches) to study the dynamics of the heme-copper oxidases and in particular to characterize specific structural/functional aspects of these dynamics. The processes at issue include: the behavior of the exogenous ligand (e.g. O2). its entrance into the protein, coordination, and activation; the behavior of the metal centers as they perform their coordination, activation, and electron transfer functions; the electron transfer reactions and the phenomena that determine their rates; the operation of the proton pump that displaces the """"""""vector protons"""""""" against the transmembrane pH gradient; the coupling of proton pumping to the electron transfer free energy; the delivery of the """"""""scalar protons"""""""", which combine with O2 to form H2O to the active site: and the structural responses of the polypeptide itself, and its sidechains, to the foregoing processes.
Our specific aims are to elucidate the following issues: details of the route of small exogenous ligands into and out of the site of O2 binding and reduction, the coordination chemistry and dynamics of the metal centers, structural features of the O2 activating site of the enzyme, the structural dynamics of the heme A prosthetic group and the Cu-B center, tests of hypotheses concerning the identity and role of residues involved in protonmotive processes, the nature of the protein dynamics which accompany (or control) the reactions of the metal centers and the protonmotive reactions, and the nature of the activation barriers involved. From this we expect to be able to infer critical new features of the functional dynamics and structure-function relationships in these systems, and to contribute to understanding of these issues in molecular detail. The results will also illuminate fundamental biophysical principles and develop experimental approaches which will be applicable to other problems in biodynamics.
