Monomers and Dimers of Cytochrome c in Apoptotic Peroxidase Activity

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This award from the Chemistry of Life Processes Program within the Chemistry Division is funding Dr. Bruce E. Bowler from the University of Montana to investigate how changes in the three-dimensional structure of a protein known as cytochrome c changes its function from a protein that moves electron around to one that speeds up the reaction of membrane lipids (fats) with hydrogen peroxide. This latter, so-called, peroxidase activity provides an early signal in important biochemical pathways. This project will also provide training for undergraduate student and graduate students in a broad array of structural, biochemical and biophysical methods. As part of this project, Dr. Bowler and his students are developing hands-on curricular materials, effective in low resource environments, to enhance STEM education at tribal schools in collaboration with the spectrUM science center in Missoula. The hypotheses for the research to be carried out derive from a novel X-ray diffraction structure of yeast iso-1-cytochrome c bound to the detergent CYMAL-6. In the presence of CYMAL-6, iso-1-cytochrome c crystallizes as a domain-swapped dimer with CYMAL-6 bound in a pocket placing its hydrocarbon chain next to the heme iron. Dr. Bowler's lab is testing the hypothesis that this dimeric structure is important for peroxidase activity by measuring the peroxidase activity of monomeric versus dimeric cytochrome c, determining the ability of cardiolipin nanodiscs to catalyze the formation of dimer, and also testing the ability of lipid analogs to stabilize the dimer. Crystallization and structural studies with different lipid analogs also is being attempted to more fully define the lipid binding pocket of the dimer. The work is being carried out with three evolutionally diverse forms of cytochrome c, those of yeast, spider monkey and human. The effects of mutations to two surface loops near the lipid binding pocket, on the accessibility of the binding pocket and the peroxidase activity of cytochrome c also is being investigated to determine how evolution from yeast to humans has optimized cytochrome c for peroxidase activity. Finally the dimeric structure of cytochrome c indicates that a broad group of lysine residues control the electrostatic binding of cytochrome c to cardiolipin. Binding studies on cytochrome c variants with these lysines eliminated is being conducted to determine the extent of the electrostatic docking site on the surface of cytochrome c.