Deciphering the Enzymatic Mechanism of Superoxide Dismutase

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From original application. No changes are being proposed. Abstract Superoxide dismutases (SODs) are the major regulators of oxidative stress and therefore the first line of defense to protect organisms against metabolic- and environmentally-induced reactive oxygen species (ROS). Human mitochondrial manganese SOD (MnSOD) expression is modulated to prevent ROS-based damage, promote redox homeostasis, and maintain proper cell signaling. Our research goal is to understand the molecular basis of how MnSOD uses coupled proton-electron transfers to dismute superoxide. For this, the 3D arrangement of all atoms is needed, most importantly the position of protons. Our recent technical advancements with neutron crystallography at Oak Ridge National Laboratory have overcome the limitations of X- ray crystallography – revealing proton positions with high detail while also allowing control of the metal electronic state. In this research project, MnSOD neutron maps will reveal the proton relays to the active site metal and the protonation states of metal- bound ligands. The scientific hypothesis for this study is that MnSOD transfers protons from a small group of water molecules via partially solvent-exposed amino acids to the nearly completely buried manganese for the dismutation of superoxide to hydrogen peroxide and molecular oxygen via cyclic metal redox reactions. The specific aims are to characterize the electron-coupled proton relays of MnSOD by investigating the proton environment of (1) the resting states of the reduced and oxidized manganese active sites, (2) the product inhibited Mn-peroxo complex, and (3) the superoxide bound enzyme. Spectroscopy on crystals will be performed to help design/understand crystallographic experiments, and computational chemistry studies on neutron derived all-atom structures will help tie the results together and test our interpretations about the enzymatic activity. The resulting protocols, methods, and structures will be of specific interest to those in the fields of structural biology, antioxidants, and metallo-enzymology and of interest to biologists in general.