Date of Graduation


Document Type


Degree Name

Bachelor of Science

Degree Level



Chemistry & Biochemistry


Millett, Frank

Committee Member/Reader

Kilyanek, Stefan

Committee Member/Second Reader

McNabb, David

Committee Member/Third Reader

Kumar, Suresh


Irreversible brain damage is commonly seen in patients that have suffered strokes, cardiac arrest, or other brain ischemia events. The hypoxic conditions result in neuron death, and previous studies have shown that additional damage occurs when blood flow is restored. It is thought that the lack of energy production during post-ischemia events also causes severe brain damage, as the brain heavily depends on oxidative phosphorylation. Cytochrome c (Cyt c) plays a crucial role in this energy production by means of the electron transport chain (ETC), transferring electrons between complexes ΙΙΙ (cytochrome bc1) and ΙV (cytochrome c oxidase, CcO). Mitochondrial release of Cyt c into the cytosol results in type ΙΙ apoptosis, and release within the mitochondria results in reduction by superoxides, suggesting that Cyt c detoxifies reactive oxygen species (ROS). It is also understood that phosphorylation of proteins within the ETC serves a regulation mechanism. We sought to determine whether the phosphorylation of a specific tyrosine (Y) residue within Cyt c, Y97, affects the binding kinetics between Cyt c and CcO, thus regulating the physiological role of Cyt c. We hypothesized that comparison of binding kinetics of mutated Y97E Cyt c and wild type Cyt c, both to CcO, in an in vitro study would reveal insight on the effects of phosphorylation at this specific residue. The Y97 residue was replaced by a glutamate (E) residue− the negative charge from E resembled the negative charge of the phosphate group in phosphorylated Cyt c. Both mutant, Y97E, and wild-type, Y97, were synthesized and purified, then subjected to analytical ultracentrifuge and laser photolysis in order to examine the binding kinetics. Results from both analytical ultracentrifuge and laser photolysis revealed little difference in binding between the mutant and the wild-type Cyt c.