Neutron diffraction analysis of human manganese superoxide dismutase

Abstract

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.

Neutron scattering in the biological sciences: progress and prospects

“…Deciphering protein chemistry will benefit from synergetic approaches combining QM/MM/MD and neutron and X-ray crystallography experiments. The most recent instruments enable data collection from crystals with unit cells of up to 250 Å (Coates et al, 2015;Azadmanesh et al, 2017). Within the next few years, systems which crystallize with large unit cells, such as protein-protein or protein-nucleic acid complexes, will become amenable to neutron crystallography, allowing overlap with cryo-EM studies of the same complexes and thereby allowing radiation-damage-free structures to be determined using neutrons, rather than electrons, as the probe.…”

Redox manipulation of the manganese metal in human manganese superoxide dismutase for neutron diffraction

“…The details of the methods for the perdeuteration, expression, purification and crystallization of MnSOD have been described previously (Azadmanesh, Trickel, Weiss et al, 2017).…”

“…In our previous work, we created a crystal system for MnSOD that was reliably applicable to neutron crystallography (Azadmanesh, Trickel, Weiss et al, 2017). The known MnSOD crystal system in a high-symmetry space group (P6 1 22) was attractive since all of the precipitating agents could be purchased in a deuterated form and complete, redundant data could be collected in a handful of data frames (as neutron beamtime is precious).…”

“…Time-of-flight information allows polychromatic diffraction patterns to be resolved into monochromatic slices, allowing the discrimination of the spatially overlapped reflections that are especially problematic for crystals with large unit cells research communications (Langan et al, 2008;Coates et al, 2010). In conjunction with perdeuteration of MnSOD and moderate crystal volumes (>0.25 mm 3 ), neutron diffraction using the MaNDi beamline was achieved to a resolution (2.14 Å ) at which deuterium positions could be observed (Azadmanesh, Trickel, Weiss et al, 2017).…”

Improving the accuracy and resolution of neutron crystallographic data by three-dimensional profile fitting of Bragg peaks in reciprocal space

“…In the case of a standard data set collected on the MaNDi beamline at the Spallation Neutron Source (SNS; Coates et al, 2015), integration errors arising from peaks near detector edges may affect as many as one fifth of the peaks. Finally, as scientifically pertinent problems continue to demand higher resolution and the analysis of larger unit cells (Azadmanesh et al, 2017), it becomes more difficult to quantify peak intensity as peaks become closer to each other and eventually overlap.…”