Presentation date: 2007-05-01Graduation date: 2007Oxygen derived radicals are involved in many aspects of life from aging and cell signaling to disease states as diverse as heart disease, diabetes, neurodegeneration and inflammation. Therefore, understanding the function of antioxidant defense proteins and the effects of oxygen derived radicals on protein function is essential to elucidate the role of reactive oxygen species in disease. This thesis describes the X-ray crystallographic structure and biochemical properties of two antioxidant defense enzymes; the N-terminal domain of alkyl hydroperoxide reductase flavoprotein (AhpF) of Salmonella typhimurium and zinc-deficient superoxide dismutase (SOD). In addition, the effect of peroxynitrite on the fluorescence of green fluorescent protein (GFP) was investigated. The N-terminal domain of AhpF has two redox active cysteines that were found to be sensitive to X-ray induced reduction. Additionally, the disulfide redox center had an unusually low redox potential in relation to the pKa of the active site thiols of other thioredoxin family members. The Nterminal domain of AhpF provides a platform to investigate the factors that govern the relationship between the pKa and reduction potential of the active site cysteines. Previous studies have shown that the loss of zinc from Cu,Zn SOD is sufficient to kill motor neurons and is important in the pathogenesis of amyotrophic lateral sclerosis. Structural studies reported herein revealed how zinc organizes the zinc-binding loop (loop IV), electrostatic loop (loop VII), and quaternary structure of SOD. The absence of zinc also increased the susceptibility of zinc-deficient SOD to aggregate in the presence of a reductant. Together these discoveries explain many of the properties that cause zinc-deficient SOD to be toxic to motor neurons. Green fluorescent protein has been proposed as a real-time marker for tyrosine nitration in vivo. We demonstrate that GFP was not sensitive enough to monitor peroxynitrite-mediated nitration in vivo, even with large bolus additions of peroxynitrite totaling 150μM. Hence, measuring the loss of GFP fluorescence in cells has limited utility as a measure of nitrative stress.