Mitochondrial cytochrome c (Cc) is a small haem protein, primarily involved in electron transfer, but is an example of an extreme multifunctional protein with roles in several stages of apoptosis through interaction with phospholipids and Apaf-1. The switch in function is associated with an interaction with cardiolipin (CL) and gain of peroxidase activity. Mutations in the Cc gene lead to Thrombocytopenia 4, a disease associated with enhanced apoptotic activity. The Y48H variant is found in the 40-57 W-loop, the lowest free-energy foldon. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes to that of the wild-type protein and G41S variant (also associated with Thrombocytopenia 4). Despite this, the intrinsic peroxidase activity is enhanced, implying that a penta-coordinate haem species is more prevalent in the Y48H variant, corroborated through determination of a Met80 off-rate of > 125 s-1 compared with ~ 6 s-1 for the wild-type protein. Heteronuclear NMR experiments with the ferric Y48H variant have revealed heighted dynamics relative to the WT protein in the 40-57 and 71-85 W-loops, the latter being the loop containing the Met80 ligand, suggesting communication between these two substructures. When these results are considered in relation to the G41S variant, a dynamic picture emerges in which heightened dynamics in key substructures of the Cc fold facilitate an increased population of a penta-coordinate, peroxidase active species of the order wild-type < G41S < Y48H. Studying the alkaline transition through pH jump stopped-flow 2 spectroscopy has revealed that not only is the pK695 is reduced by ~ 1 pH unit compared to that of the wild-type protein, but also the pK of the trigger is lowered. In contrast, variants in the 71-85 W-loop do not alter the pK of the trigger but are nevertheless associated with enhanced peroxidase activity relative to the wild-type protein, due to enhanced local dynamics within the haem crevice.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:743960 |
| Date | January 2018 |
| Creators | Deacon, Oliver Mario |
| Publisher | University of Essex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://repository.essex.ac.uk/22285/ |
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