published_or_final_version / Chemistry / Master / Master of Philosophy

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Cytochrome bc1 (Complex III) is an important enzyme that takes part in the respiratory electron transport chain in vertebrates, yeast, and many bacteria. The complex exists as a dimer, in which each monomer contains three catalytic subunits: cytochrome c1, cytochrome b and the Rieske iron-sulfur protein or ISP. Within the inner mitochondrial membranes of eukaryotes, Complex III catalyzes the transfer of two electrons from ubiquinol (UQH2) to cytochrome c, a water-soluble protein, through a process called the modified Q-cycle mechanism. Under very specific conditions, such as mutations within cytochrome b, disruption of the normal mechanism leads to bypass reactions, including the formation of superoxide and reactive oxygen species. We have sought to restore the activity of a mutant of cytochrome b (T61V) by modifying UQH2, the natural substrate for this enzyme. The structure of the oxidized form, UQ consists of a p-quinone head group and a hydrophobic all-trans polyprenyl unit (tail) that can vary in length, depending on the species in which it is found. The present work highlights modifications to the substituent groups attached to the quinone head and to the all-trans polyprenyl tail. Since the midpoint potentials of these molecules are pH dependent, cyclic voltammetry and spectroelectrochemistry studies in buffered aqueous solutions have been carried out on these molecules (analogs of UQ10). Modifications of the substituent groups attached to the quinone head gave the molecules a different ability to either donate or receive electrons, while modifications to the length of the tail either increased or decreased the solubility of these molecules inside the phospholipid membrane. We examined the normal activity and the production of superoxide in wild-type and (T61V) of bacterial Rhodobacter sphaeroides in the presence of these analogs. We confirmed that to prevent damaging side reactions, normal operation of the Q-cycle requires a fairly narrow window of reduction potentials with respect to the ubiquinol substrate.

Heart transplantation is often the only therapeutic option for patients with end stage heart disease. Allograft organs are in short supply. Thus, preserving the life of a grafted organ is extremely important. Cardiac allograft vasculopathy (CAV) is an expression of chronic rejection that accounts for the greatest loss of graft function in transplanted hearts. Peri-transplant ischemia/reperfusion (I/R)-injury occurs during transplantation when blood flow is stopped to remove the heart from the donor and then is reinstated upon implantation of the donor heart into the recipient. This oxidative injury contributes to vascular dysfunction and CAV. In this dissertation, I hypothesize that prevention and/or reduction of I/R during transplantation reduces post-transplant vascular dysfunction and CAV. In this regard, myself and my colleagues examined the roles of apoptosis repressor with caspase recruitment domain (ARC) and cytochrome p450 (CYP) 2C enzymes in UR-induced vascular dysfunction and CAV. ARC expression was detected in endothelial cells (ECs) and smooth muscle cells (SMCs); however, increased levels of ARC do not protect against oxidant injury. ARC overexpression did protect against oxidant-induced cell death in H9c2 rat embryonic myoblasts. We observed that ARC-overexpression prevented H9c2 differentiation into muscle cells. With our focus on vascular injury, we turned our attention to the CYP 2C enzymes. Both endothelium-dependent and independent vascular function was impaired following I/R. Pre-treatment with the CYP 2C inhibitor sulfaphenazole (SP) restored endothelial sensitivity to acetylcholine, but did not restore sensitivity to endothelium-independent vasodilators. Rat heterotopic heart transplants were performed with rats being treated with SP or vector control prior to surgery. Rats treated with SP showed significantly reduced luminal narrowing and had decreased SMC proliferation, oxidant and interferon-y levels. No differences were detected in immune infiltration or apoptosis. Complementary studies in cultured vascular cells revealed that CYP 2C9 expression decreased viability and increased ROS production following hypoxia and re-oxygenation in ECs but not in SMCs. In summary, we did not detect protection of vascular cells by ARC, but did discover a novel role for ARC in differentiation. CYP 2C contributes to post-ischemic vascular dysfunction and CAV through increased oxidative stress and endothelial dysfunction.

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