With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Drs. Denis Rousseau, Syun-Ru Yeh and Gary Gerfen from the Albert Einstein College of Medicine to quantitatively determine the factors that determine how the reduction of oxygen to water is coupled to proton translocation in cytochrome c oxidase. Over 95% of the oxygen consumed in humans is taken up by cytochrome c oxidase, an enzyme located in the inner membrane of mitochondria. The reduction of oxygen to water by the enzyme generates energy that is harnessed to translocate protons across the inner mitochondrial membrane, forming a proton gradient, which in turn is used to generate chemical energy for use in the cell. A major gap in our understanding is how the oxygen reduction is coupled to the proton translocation. In this grant a postulated mechanism for the coupling will be explored. The results of this project are expected to yield new insights into the functional properties of cytochrome c oxidase, and thereby impact the field of Bioenergetics. The experiments will involve both the development of new rapid mixing techniques and the use of state-of-the-art spectroscopic technology, which will serve as an outstanding training platform for students and postdoctoral fellows.
Previous studies have shown that the reaction of hydrogen peroxide with cytochrome c oxidase generates many of the same intermediates as those formed in the reaction of oxygen with the cytochrome c oxidase. The mechanism by which hydrogen peroxide reacts with cytochrome c oxidase is unknown. The hypothesis that hydrogen peroxide reacts with the heme a3 group and forms a radical species as the first step in the reaction and that the radical subsequently migrates to amino acid residues will be tested. A proposed model for proton translocation based on the transfer of a radical from Tyr244 to Tyr129 will be tested also. The pH, the hydrogen peroxide concentration, and the oxygen concentration will be varied to determine how the reaction is regulated. The measurements will identify where the initially formed radical is located and the time dependence of the various intermediates that are subsequently formed. Finally, the full evolution of the intermediates into those detected in the past by hand mixing techniques will be determined. Rapid mixing techniques will be used to initiate the reaction, rapid freeze quench methods will be used to trap the intermediates, and new silicon mixers will be designed and fabricated; these methods will make possible the following up of the reaction on timescales from microseconds to minutes. EPR spectroscopy will be used to study the intermediates and reaction products. Students and post-doctoral fellows who participate in the project will gain education and training in Bioenergetics, advanced technology development and state-of-the-art spectroscopy. New silicon-based mixers designed for this project will be made available to the wider academic community.
