Cryotrapping peroxide in the active site of human mitochondrial manganese superoxide dismutase crystals for neutron diffraction

Abstract

Structurally identifying the enzymatic intermediates of redox proteins has been elusive due to difficulty in resolving the H atoms involved in catalysis and the susceptibility of ligand complexes to photoreduction from X-rays. Cryotrapping ligands for neutron protein crystallography combines two powerful tools that offer the advantage of directly identifying hydrogen positions in redox-enzyme intermediates without radiolytic perturbation of metal-containing active sites. However, translating cryogenic techniques from X-ray to neutron crystallography is not straightforward due to the large crystal volumes and long data-collection times. Here, methods have been developed to visualize the evasive peroxo complex of manganese superoxide dismutase (MnSOD) so that all atoms, including H atoms, could be visualized. The subsequent cryocooling and ligand-trapping methods resulted in neutron data collection to 2.30 Å resolution. The P6122 crystal form of MnSOD is challenging because it has some of the largest unit-cell dimensions (a = b = 77.8, c = 236.8 Å) ever studied using high-resolution cryo-neutron crystallography. The resulting neutron diffraction data permitted the visualization of a dioxygen species bound to the MnSOD active-site metal that was indicative of successful cryotrapping.

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition

“…We pursued the neutron structure we to visualize a paramagnetic center-oxygen species interaction without radiation-induced perturbations 33 and identify all the proton positions in the active site. We strategically chose the variant to study product inhibition due to the propensity to enrich and retain the product-inhibited complex at full occupancy without perturbing the hydrogen bond network 26,30,45,46,51 . First, we solved the all-atom structure of the entire enzyme excluding the active site.…”

“…1) and a previous peroxidesoaked Escherichia coli wildtype MnSOD structure. However, the dioxygen molecules in these X-ray structures are slightly different in orientations and are at partial occupancies probably due to the detrimental effects of Xray irradiation 51,52 . At physiological temperatures, optical absorption spectra also suggest a displacement of WAT1 and binding of a dioxygen species, which agrees with our data even though the ligand is cryotrapped 53 .…”

“…The 4 M deuterated potassium phosphate also served as the cryoprotectant for the cryocooling process. Further details of the process were published 51 . For the crystals grown on earth, the initial deuterium exchange of crystals was performed by vapor diffusion in quartz capillaries using deuterated solutions of 2.3 M deuterated potassium phosphate pD 7.8.…”

“…Flash-cooling was performed with an Oxford diffraction cryostream 77 . Further details of ligand cryotrapping were published 51 .…”

Perfect Crystals: Microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography

“…An atomic understanding of MnSOD catalysis requires direct proton visualization, which can be achieved by neutron crystallography 2,3 . The crystals of MnSOD grown in microgravity on the International Space Station (ISS) have enabled the collection of highresolution neutron diffraction 4 and will make possible the determination of several neutron structures.…”