Conformational diversity of cytochrome C on cardiolipin-containing liposomes probed by optical spectroscopy

Leah Angelina Pandiscia

doi:10.17918/etd-6599

For over 60 years, cytochrome c has served as a model system for protein folding and electron transfer studies. In spite of adopting a rather rigid structure around its prosthetic heme group, it is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria, cytochrome c, in its native state, is involved in the electron transport chain where it shuttles electrons between cytochrome c reductase and cytochrome c oxidase to facilitate the production of adenosine triphosphate (ATP). Recently, cytochrome c has been identified as a trigger of mitochondrial apoptosis. To this end, the protein must dissociate from the inner mitochondrial membrane in order to be released into the cytosol where it binds to apoptotic complexes. In order to perform this task the protein must first gain peroxidase activity in order to trigger the apoptotic pathway which requires a conformational transition. To explore the conformational manifold that cytochrome c can adopt on membrane surfaces that allows the protein to function as electron carrier and as peroxidase, we combined absorption, visible circular dichroism and fluorescence spectroscopy to probe the response of specific structural regions of the protein to the protein's binding to cardiolipin-containing liposomes. The latter have served as ideal model systems to investigate the various modes of interaction between cytochrome c and the inner mitochondrial membrane. The binding of cytochrome c to cardiolipin-containing liposomes was examined as a function of cardiolipin (CL)/phosphocholine (PC) mixtures, cardiolipin concentration and the concentration of NaCl, which has the capacity to inhibit binding governed by electrostatic interactions. It was the purpose of these studies to obtain a thorough thermodynamic and spectroscopic characterization of cytochrome c binding to cardiolipin-containing liposomes at conditions which, in terms of cardiolipin fraction and salt concentration, bear some similarity to what the protein encounters on the inner membrane of the mitochondria. From our experimental data sets, we were able to model our system, described by the coexistence of two conformations: one that is unfolded and has a high fluorescence yield while the other is very native-like but not identical to native state. Here, the M80 is still ligated and the protein has an intact heme pocket. Only the later state can perform electron transfer. The former state could be candidate for peroxidase activity.

Conformational diversity of cytochrome C on cardiolipin-containing liposomes probed by optical spectroscopy

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Conformational diversity of cytochrome C on cardiolipin-containing liposomes probed by optical spectroscopy

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