Oxidative stress is caused by an imbalance between production and accumulation of oxygen reactive species (ROS) in cells and tissues. Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme that protects the mitochondria from oxidative damage by using a mechanism involving electron and proton transfers to lower ROS levels in mitochondria. More than a quarter of known enzymes also rely on electron and proton transfers to facilitate cellular activities that are essential for human health. However, most of their mechanisms are unclear. Now researchers from the University of Nebraska Medical Center (UNMC) and the Department of Energy’s (DOE’s) Oak Ridge National Laboratory (ORNL) report they have now observed the complete atomic structure of MnSOD, including its proton arrangements, with neutron scattering.

Their findings were published in the journal Nature Communications in a paper titled, “Direct detection of coupled proton and electron transfers in human manganese superoxide dismutase.”

“Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix,” wrote the researchers. “Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O2•−. The mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn3+ and Mn2+ enzyme forms.”

Neutron diffraction or elastic neutron scattering is the application of neutron scattering to the determination of the atomic and/or magnetic structure of a material.

“Using neutrons, we were able to see MnSOD features that were completely unexpected, and we believe this will revolutionize how people think this enzyme and other enzymes like it operate,” explained Gloria Borgstahl, PhD, a UNMC professor and corresponding author of the study.

Previously, the enzyme’s sequence of electron and proton transfers had not been defined at the atomic level because of challenges in tracking how protons are shuttled between molecules. A fundamental understanding of this catalytic process could inform therapeutic approaches that harness this enzyme’s antioxidant abilities.

“Because neutrons are particles that do not interact with charge, they don’t interfere with the electronic properties of metals, which makes them an ideal probe for analyzing metal-containing enzymes, like MnSOD,” added Leighton Coates, PhD, an ORNL neutron scattering scientist involved with this study. “Additionally, neutrons don’t cause radiation damage to materials, allowing us to collect multiple snapshots of the same sample as it shifts between electronic states.”

Using MaNDi, the macromolecular neutron diffractometer at ORNL’s Spallation Neutron Source (SNS), the researchers were able to map out the entire atomic structure of MnSOD and observed how the enzyme’s protons change when it gains or loses an electron. By analyzing the neutron data, the scientists traced the pathways of protons as they moved around the active site.

Their findings suggest that catalysis involves two internal proton transfers between the enzyme’s amino acids and two external proton transfers that originate from solvent molecules. Their findings confirmed some past predictions of the enzyme’s biochemical nature, but to their surprise, they also found some unexpected features.

“Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond,” noted the researchers.

Their findings demonstrate that this mechanism is more complex than previously believed.

Moving forward, the researchers will next examine the enzyme’s structure when it is bound to a superoxide substrate. They also plan to observe mutated components of MnSOD and neutron analysis to other enzymes.

“Over a fourth of all known enzyme activities involve electron and proton transfers,” said Jahaun Azadmanesh, a PhD candidate and researcher at UNMC and study co-author. “MnSOD is just one enzyme in a sea of many others, and with neutrons, we can study their catalytic mechanisms to a level of detail that hasn’t been possible before.”

These findings can provide insights into additional mechanisms of this antioxidant enzyme and may lead to novel strategies to fight against diseases associated with defective MnSOD activity.