Abstract
Cytochrome c is a highly conserved, haem containing protein that usually resides in the mitochondria inter-membrane space. In mitochondria, cytochrome c has a well-known role in the electron transport chain transferring electrons from complex three to complex four. Cytochrome c also has an important role in the intrinsic apoptosis pathway. Following an apoptotic trigger, cytochrome c is released from mitochondria into the cytosol where it triggers apoptosome formation by binding to apoptosis protease activating factor-1 (Apaf-1). Apoptosome formation leads to the activation of cysteine-aspartic proteases (caspases) and cell death. Prior to release from mitochondria, cytochrome c is reported to catalyse the oxidation of cardiolipin in the presence of H2O2. This peroxidase reaction has been said to be necessary for cytochrome c release from mitochondria.
Three pathogenic variants in cytochrome c have been reported in families with thrombocytopenia, characterised by a low platelet count in the blood. These variants increase the peroxidase activity of cytochrome c in vitro with a non-physiological substrate. The first aim of the work described in this thesis was to determine the underlying mechanism behind this increase in peroxidase activity. It was hypothesised that the mutations alter the protein dynamics of cytochrome c, leading to the increase in activity. However, the results showed that the mutations had no effect on the protein dynamics of cytochrome c throughout the steady state of the peroxidase reaction. The Km for a non-physiological substrate was also determined however, unexpectedly the rate of the peroxidase reaction decreased with increasing substrate concentration. It was concluded that some substrates can inhibit the initial oxidation of methionine 80 in cytochrome c, a step that is required for the enzyme to have peroxidase activity. This may have implications in the cell as small molecules could inhibit the peroxidase reaction in vivo.
Mutations in cytochrome c also increase the activation of caspases in vitro. It was hypothesised that cytochrome c mutations alter the affinity of the interaction between cytochrome c and Apaf-1, causing the increase in caspase activation. However, due to Apaf-1 oligomerisation following cytochrome c binding, the binding affinity is hard to measure with accuracy. As cytochrome c binds to the WD40 domain of Apaf-1, we attempted to express the WD40 domain to use when determining the binding affinity. This was unsuccessful due to multiple WD40 domain constructs being insoluble following expression in Escherichia coli and insect cells. Using full-length Apaf-1 to determine the binding affinity was attempted using BLItz. This was also unsuccessful due to non-specific binding to several types of biosensor. Alternative methods will need to be tried to express the Apaf-1 WD40 domain and determine the affinity of the interaction.