ORCID ID

0000-0002-3615-7785

Graduation Date

Fall 12-18-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Biochemistry & Molecular Biology

First Advisor

Gloria Borgstahl

Second Advisor

Justin Mott

Third Advisor

Rebecca Oberley-Deegan

Fourth Advisor

Tahir Tahirov

Abstract

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of human SODs that dwells within the mitochondria and is responsible for protecting the organelle against superoxide-mediated damage. The protein is arguably the most significant antioxidant enzyme as the mitochondria are especially integral for cellular vitality. This is exemplified by the embryonic lethality of mice lacking MnSOD and the multitude of human disease states that manifest as a result of dysfunctional MnSOD. The bioprotective attributes of MnSOD have attracted the attention of clinicians and is illustrated by the multiple ongoing clinical trials that attempt to mimic the function of the enzyme. While MnSOD has proven to be of significant importance for human vitality and has been studied extensively since its discovery over 50 years ago, its atom-by-atom mechanism has still been elusive and the mechanism of MnSOD has yet to be defined due to its nature of catalysis. MnSOD performs its function through concerted proton-electron transfers (CPETs) at specific sites of the enzyme that have been extremely difficult to detect experimentally. An emerging biophysical tool capable of circumventing previous experimental obstacles is neutron protein crystallography. This method involves diffracting neutrons off of crystallized protein samples with controlled electronic states into a pattern that can be deciphered for specific proton sites thereby permitting the experimental coupling of proton and electron transfers. In this thesis work, significant revelations are made for the mechanism of MnSOD using a multitude of approaches, including neutron crystallography where significant developments are also made for the emerging technique.

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