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

Jahaun Azadmanesh, William E. Lutz, Leighton Coates, Kevin L. Weiss, Gloria E.O. Borgstahl

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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.

Original languageEnglish
Pages (from-to)8-16
Number of pages9
JournalActa Crystallographica Section F:Structural Biology Communications
Volume78
DOIs
StatePublished - Jan 1 2022

Funding

This research was supported by NASA EPSCoR (NE-80NSSC17M0030 and NE-NNX15AM82A). The UNMC Structural Biology Core Facility was funded by the Fred and Pamela Buffett NCI Cancer Center Support Grant (P30CA036727). LC acknowledges support from the NIH (R01-GM071939). The research at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source was sponsored by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. The Office of Biological and Environmental Research supported research at ORNL Center for Structural Molecular Biology (CSMB) using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. LC acknowledges support from the Second Target Station, a DOE Office of Science User Facilities Construction Project at Oak Ridge National Laboratory.

FundersFunder number
Fred and Pamela Buffett NCIP30CA036727
NASA EPSCoRNE-80NSSC17M0030, NE-NNX15AM82A
Scientific User Facilities Division
Second Target Station
National Institutes of HealthR01-GM071939
U.S. Department of Energy
National Institute of General Medical SciencesR01GM145647
Office of Science
Basic Energy Sciences
Biological and Environmental Research
Oak Ridge National Laboratory

    Keywords

    • Cryotrapping
    • Human manganese superoxide dismutase
    • Large unit cell
    • Neutron diffraction
    • Peroxide

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