Intellectual Merit: Cytochrome c is a multifunctional protein, localized in the intermembrane space of mitochondria. A primary function of cytochrome c is as an intermediary in the electron transport chain where it aids in the production and storage of the energy needed for life. It also acts as a signaling agent in the programmed cell death pathway, apoptosis, an important pathway in the growth and development of complex organisms. To carry out both functions, cytochrome c must convert back and forth between two different structures. In the structure used for electron transport, cytochrome c has a methionine amino acid at sequence position 80 (Met80) bound to the iron atom of the heme cofactor of the protein. In the early stages of apoptosis, cytochrome c acts as a peroxidase. In this role, it catalyzes the addition of oxygen to a lipid known as cardiolipin, which is located in the inner membrane of mitochondria. To catalyze this reaction, the interaction between the Met80 and the iron must be disrupted to allow oxygen or peroxide to bind to and be activated by the iron of the heme cofactor. The primary focus of this project has been to study the structures of cytochrome c that mediate peroxidase activity and to test several hypotheses about how the sequence of amino acids that make up cytochrome c has evolved to optimize the protein to carry out peroxidase activity.

This laboratory has made a number of variant forms of cytochrome c with amino acid substitutions near Met80 in the sequence of the protein. Studies on these protein variants show that amino acids at sequence positions 72, 81 and 83 all impact the ability of cytochrome c to convert from the structure that mediates electron transfer to the structure that mediates peroxidase activity. Studies on the peroxidase activity show that the level of activity correlates well with the ability of the protein to convert to a structure that is competent for peroxidase activity. The laboratory has also characterized the binding of cytochrome c to membrane models containing cardiolipin, known as liposomes. These studies show that cytochrome c undergoes two stages of structural rearrangement on the surface of the liposome that depend on the available space on the membrane surface. To better understand the nature of the interaction between cytochrome c and cardiolipin containing membranes, crystals of cytochrome c in the presence and absence of detergent molecules (as mimics of cardiolipin) were grown. X-ray diffraction methods were applied to these crystals to obtain atomic resolution structures of two alternate conformers of cytochrome c. Both of these structures no longer have Met80 bound to the iron atom of the heme cofactor. In one structure, a detergent molecule is bound in a channel that places the end of the hydrocarbon chain of the detergent near the heme iron, poised to be oxygenated by oxygen or peroxide bound to the iron atom of the heme cofactor (see the image of this structure). The results of this project have been disseminated to the scientific community through nine papers published in scientific journals, with one additional manuscript now under consideration for publication. The PI and the graduate students working on this project have made 25 presentations on the results from this project at scientific meetings and at universities.

Broader Impacts: Three undergraduate and four graduate students have received training in basic molecular biology, protein expression and purification, and biophysical and structural biology methods necessary to characterize the structural and functional properties of alternate conformers of cytochrome c. These students have also developed the writing and presentation skills necessary to communicate their science to the scientific community. One graduate student has completed his PhD during the project period and another has completed her MS degree. A third graduate student will complete her PhD in the next 6 months. One of the undergraduate students is currently in medical school and another is pursuing an MS degree. Thus, significant human resource development of scientific professionals has been supported by this grant. The grant has also supported an outreach project for the spectrUM Discovery Area science museum in Missoula, Montana. The laboratory has developed materials, which are displayed through an interactive application loaded onto iPads. The iPads are available in the entry area of the museum. The iPad application introduces the relationship between protein structure and function. The application makes extensive use of three-dimensional graphics to illustrate how the shapes of proteins change as they carry out their functions. The primary audience for these materials are K-12 students with the intent of sparking their interest in pursuing careers in science.

Last Modified: 12/13/2017
Modified by: Bruce E Bowler