Jahaun Azadmanesh, University of Nebraska Medical Center

Abstract

Understanding Proton Coupled Electron Transfer in Human Manganese Superoxide Dismutase

Jahaun Azadmanesh1, Katelyn Slobodnik1, William E. Lutz1, Leighton Coates2, Kevin L. Weiss3, Dean A. A. Myles3, Thomas Kroll4, Rebecca Oberley-Deegan5, and Gloria E. O. Borgstahl1*

1Eppley Institute for Cancer and Allied Diseases, 986805 Nebraska Medical Center, Omaha, NE 68198-6805, USA
2Second Target Station, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA
3Neutron Scattering Division, Oak Ridge National Laboratory, 1 Bethel Valley Road, Oak Ridge, TN 37831, USA
4Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, USA
5Department of Biochemistry and Molecular Biology, 985870 Nebraska Medical Center, Omaha, NE 68198-5870, USA
*gborgstahl@unmc.edu

About 25% of known enzymes are oxidoreductases that catalyze the electron transfers that life depends on. To chemically accelerate redox reactions to the rates needed for life, oxidoreductases couple the transfer of electrons to the transfer of protons in a process called proton-coupled electron transfer (PCET). The biochemistry behind enzymes utilizing PCETs is not well understood as it requires precise definition of the proton donors/acceptors in tandem with electron transfer steps. Of particular note are oxidoreductases that regulate the concentration of reactive oxygen species (ROS) in cells through PCET-mediated redox reactions. ROS levels mediate mitophagy and programmed cell death and dysfunction of oxidoreductases responsible for limiting ROS concentrations contribute to cardiovascular disease, neurological disease, and cancer progression.
Human manganese superoxide dismutase (MnSOD) is an oxidoreductase found in the mitochondrial matrix that decreases O2●- concentrations using PCET reactions. MnSOD eliminates O2●- by oxidation to O2 with a trivalent Mn ion (k1), and reduction to H2O2 with a divalent Mn ion (k2). MnSOD is the only means the mitochondrial matrix has to limit O2●- levels low enough to avoid damage to macromolecules and is a central axis to preserving mitochondrial function.

Mn3+ + O2•- ↔ Mn2+ + O2 k1 = 1.5 nM-1 s-1
Mn2+ + O2•- + 2H+ ↔ Mn3+ + H2O2 k2 = 1.1 nM-1 s-1

Here, we seek to obtain a full understanding the MnSOD PCET mechanism by obtaining an electronic description of the redox center with X-ray absorption spectroscopy (XAS) using the beamlines of the Stanford Synchrotron Radiation Lightsource (SSRL) and coupling it with knowledge of the protonation states at the active site found through neutron diffraction at Oak Ridge National Laboratory (ORNL).

For each enzymatic step, we sought to obtain data on the metal-ligand distances, the oxidation state of the Mn metal, and the metal coordination environment using the XAS techniques of extended X-ray absorption fine spectra (EXAFS) and high energy resolution fluorescence detected X-ray absorption near edge structure (HERFD-XANES). HERFD-XANES also yields a pre-edge signal that originates from 1s → 3d transitions with an increase in intensity upon 4p character mixing into the 3d orbitals. These data are complemented with EXAFS simulations utilizing FEFF to fit a geometric model and time-dependent density functional theory (TD-DFT) calculations to enhance spectral assignment of pre-edge intensities. Overall, investigating the electronic details of the Mn metal at the active site of the MnSOD enzyme provides essential information towards how PCET catalysis occurs.