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Mitochondrial membrane mimic-induced structural dynamics of cytochrome c as probed by vibrational spectroscopy
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Mitochondrial membrane mimic-induced structural dynamics of cytochrome c as probed by vibrational spectroscopy

Dmitry Malyshka
Master of Science (M.S.), Drexel University
Jun 2017
DOI:
https://doi.org/10.17918/etd-7423
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Abstract

Cardiolipin Cytochrome c Infrared spectroscopy Raman spectroscopy Apoptosis
Cytochrome c has been widely studied as an electron transport protein due to its main function as an electron shuttle in the mitochondria. It performs this role via its native methionine/histidine ligation of the heme group, and ultimately drives ATP formation by indirectly pumping protons across the inner mitochondrial membrane to form a gradient. However, more recently, it was shown that cytochrome c plays a rather large part in apoptosis, a role it must play though the gaining of peroxidase activity on the surface of the cardiolipin-containing membrane (inner mitochondrial membrane). How does it balance the two functions? Binding studies have now shown that there are multiple binding sites (A, C, and L). Furthermore, some binding models have been proposed as well that treat the binding as a CL-dependent conformational equilibrium between native-like and non-native like states. However, the question as to what the exact structural configuration of these states is still outstanding, along with the unresolved issue of cardiolipin headgroup ionization. Though infrared spectroscopy experiments, it was shown that CL is doubly ionized at neutral pH, with DFT calculations aimed at aiding in the analysis and assignments of phosphate group infrared specra. With resonance Raman, a new experimental strategy is introduced where photoreduction can be used to trace the amount of the non-native-like bound state lacking the native methionine ligation. Furthermore, when the binding was measured at the biologically relevant slightly acidic pH, distinct evidence for the presence of high-spin was observed. Clearly, these states are the prime candidates for peroxidase activity. Furthermore, the different mode of binding observed at slightly acidic pH is most likely due to the protonation of one of the histidine residues that is responsible for replacing the native methionine during CL-induced unfolding.

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