Superoxide dismutases (SODs) are enzymes that play a key role in protecting cells from toxic oxygen metabolites by disproportionation of two molecules of superoxide into molecular oxygen and hydrogen peroxide via cyclic reduction and oxidation at the active site metal. The azide anion is a potent competitive inhibitor that binds directly to the metal and is used as a substrate analog to superoxide in studies of SOD. The crystal structure of human MnSOD-azide complex was solved and shows the putative binding position of superoxide, providing a model for binding to the active site. Azide is bound end-on at the sixth coordinate position of the manganese ion. Tetrameric electrostatic surfaces were calculated incorporating accurate partial charges for the active site in three states, including a state with superoxide coordinated to the metal using the position of azide as a model. These show facilitation of the anionic ligand to the active site pit via a ‘valley’ of positively-charged surface patches. Surrounding ridges of negative charge help guide the superoxide anion. Within the active site pit, Arg173 and Glu162 further guide and align superoxide for efficient catalysis. Superoxide coordination at the sixth position causes the electrostatic surface of the active site pit to become nearly neutral. A model for electrostatic-mediated diffusion, and efficient binding of superoxide for catalysis is presented.

Superoxide dismutases (SODs) are enzymes that protect against oxidative stress by dismutation of superoxide into oxygen and hydrogen peroxide through cyclic reduction and oxidation of the active-site metal. The complete enzymatic mechanisms of SODs are unknown since data on the positions of hydrogen are limited. Here, methods are presented for large crystal growth and neutron data collection of human manganese SOD (MnSOD) using perdeuteration and the MaNDi beamline at Oak Ridge National Laboratory. The crystal from which the human MnSOD data set was obtained is the crystal with the largest unit-cell edge (240 Å ) from which data have been collected via neutron diffraction to sufficient resolution (2.30 Å ) where hydrogen positions can be observed.

Superoxide dismutases (SODs) are necessary antioxidant enzymes that protect cells from oxidative stress by converting superoxide, a reactive oxygen species, into molecular oxygen and hydrogen peroxide via cyclic reduction and oxidation at the active site metal. Oxidative stress is involved in many disease states, including cancer, neurological disorders, and heart disease. Despite their protective biological importance, the complete multistep enzymatic mechanisms of SODs are unknown due to limitations in identifying the positions of hydrogen atoms at the active site. Knowledge of the hydrogen positions are critical because (1) they are needed to differentiate the ligands in the active site, which are hypothesized to differ based on the redox state of the active site metal, and (2) they reveal the source and pathway of protons to the catalytic center for proton-assisted electron transfer.The missing structural data on SOD complexes and intermediates can be revealed using Neutron Macromolecular Crystallography (NMC). Neutron diffraction has multiple advantages over X-ray diffraction. (1) Neutrons probe structure without radiation damage, allowing high quality data collection at room temperature. (2) They are inert and do not reduce metals like Xrays do, allowing crystallographic studies on fully oxidized metalloproteins. (3) Hydrogen placement can be identified without the need of < 1 Å data that X-ray diffraction requires. Only ~0.5% of Protein Data Bank (PDB) X-ray crystal structures have high enough resolution to identify hydrogen atoms. Neutron diffraction requires 2.50 Å data or better. The limiting factor for NMC is the requirement for large crystals and/or deuteration of the sample. Consequently, neutron diffraction will be vital for elucidating the catalytic mechanism of SODs due to their dependence on redox state and exchangeable hydrogens to facilitate catalysis.

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