Deciphering the Enzymatic Mechanism of Superoxide Dismutase

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.

Grant Number: 5R01GM145647-04
NIH Institute/Center: NIH
Principal Investigator: Gloria Borgstahl