OXYGEN UTILIZING MEMBRANE HEME PROTEINS

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The goal of this Program Project entitled Oxygen Utilizing Membrane Heme Proteins is to characterize the structures of prostaglandin H synthases (GHSs) and cytochrome c oxidase (CcOX) in the context of the chemical changes that occur during catalysis as these enzymes interact with their substrates and with biological membranes. PGHS catalyzes the initial step in the biosynthesis of prostanoids--the formation of prostaglandin endoperoxide H2 from arachidonic acid, two molecules of 02 and two electrons. CcOX is a terminal heme/Cu oxidase of the respiratory electron- transfer chain which catalyzes a net four electron reduction of 02 to two H20 molecules with concomitant translocation of four protons across the mitochondrial inner membrane (or bacterial plasma membrane). There are five projects and four cores. Project I: Cyclooxygenase Catalysis and Suicide Inactivation (Smith) will examine the binding of arachidonate to the cyclooxygenase site of PGHS, the mechanism of suicide inactivation and the role of H20 channels in PGHS. Project II: Structural Biology of Peroxidation by PGH Synthases (Garavito) will examine the structural aspects of peroxidase catalysis in PGHSs and discern the structural basis for the differences in the peroxidative activities of PGHS-1 and -2. Project III: Monotopic Membrane Anchors in PGH Synthases-1 and -2 (DeWitt) will test the hypothesis that PGHS-1 and PGHS-2 associate with a single leaflet of the membrane bilayer through hydrophobic faces of four contiguous amphipathic helices present in the amino-terminal third of the proteins. Project IV: Substrate Docking in Cytochrome c resolved Spectroscopy of Cytochrome Oxidases and PGH Synthases (Babcock) is designed to understand oxygen and peroxide activation by CcOX and PGHSs and the mechanisms by which the free energy is released in the reduction of these substrates. Core A. Administration (Smith), Core B: Membrane Protein Expression and Purification (DeWitt), Core C: Crystallization and X-ray Crystallography (Garavito) and Core D: EPR and Resonance Raman Spectroscopy (McCracken) provide administrative and technical support for these projects.