Abstract
The textbooks of biochemistry have painted a picture of cytochrome c only being involved in the electron transfer chain for 100 years, but the mitochondrial haem protein also plays important roles in the intrinsic apoptosis pathway. Cytochrome c acquires a new function during apoptosis; peroxidase activity, leading to peroxidation of cardiolipin (a mitochondrial phospholipid) which is proposed to be a required step in the intrinsic apoptosis pathway. The overall aim of this thesis was to better understand the peroxidase function of cytochrome c by studying mutant forms of the protein.
Mutations of cytochrome c causes thrombocytopenia (low platelets). The molecular mechanism underlying the phenotype is unknown. Three cytochrome c mutations have been reported, G41S, Y48H, and A51V. All mutations occur in a surface Ω loop (residues 40-57). Our lab has previously reported that the naturally occurring G41S cytochrome c and a recombinant G41T cytochrome c variants have increased peroxidase activity. Additionally, newly identified naturally occurring mutations, Y48H and A51V cytochrome c, also manisfested increased peroxidase activity. The underlying mechanism of increased peroxidase activity is unknown.
This thesis aimed to investigate the peroxidase activity of cytochrome c by addressing two aims. Firstly, by understanding how the mutations of cytochrome c alters the peroxidase activity. Secondly, by determing the relationship between the peroxidase activity and cardiolipin oxidation. Native cytochrome c is a poor peroxidase because its iron atom is hexacoordinated. It is proposed that loss of the coordination between the iron atom and Met80 is necessary for the commencement of the peroxidase activity. Absorbance spectroscopy, mass spectrometry and high-performance liquid chromatography techniques were used to monitor the kinetic behaviour of the haem coordination of Gly41 cytochrome c variants during the peroxidase reaction. The peroxidase reaction catalysed by the cytochrome c variants consists of a slower initial rate followed by a faster steady state rate. The difference between the initial and steady state was significantly greater for the mutations of cytochrome c. Our results demonstrate that mutation of residue 41 to Thr or Ser resulted in a specific oxidation of Met80 with the amount of Met80-SO species formed is consistent with the impact of each mutation on the peroxidase reaction rate (G41T>G41S>WT).
The interaction of cytochrome c with cardiolipin in liposomes causes a conformational change in cytochrome c, stimulating its peroxidase activity. Our results show that the cytochrome c variants differ in their ability to bind to cardiolipin-containing liposomes and mitoplasts, resulting in differences in cardiolipin-dependent stimulation of peroxidase activity. Moreover, using mass spectroscopy to analyse lipid oxidation, cytochrome c-catalysed oxidation of cardiolipin was dependent on liposome composition and on the reaction conditions. In addition, when cytochrome c-depleted mitoplasts were used, minimal cardiolipin oxidation was detected. Although the cytochrome c variants are a better peroxidase under in vitro conditions, the relevance of this to cardiolipin oxidation in vivo during apoptosis is still questionable. Thus, the results described in this thesis opened an interrogative role of cytochrome c as a peroxidase in a mitochondrial environment.