Title: 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 P 6 ₁ 22 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.

Authors:

Azadmanesh, Jahaun ;

• Search DOE PAGES for author "Azadmanesh, Jahaun"
• Search DOE PAGES for ORCID "0000-0002-3615-7785"

Lutz, William E. ;

• Search DOE PAGES for author "Lutz, William E."
• Search DOE PAGES for ORCID "0000-0002-7248-4994"

Coates, Leighton ;

• Search DOE PAGES for author "Coates, Leighton"
• Search DOE PAGES for ORCID "0000-0003-2342-049X"

Weiss, Kevin L. ;

• Search DOE PAGES for author "Weiss, Kevin L."
• Search DOE PAGES for ORCID "0000-0002-6486-8007"

Borgstahl, Gloria E. O.

• Search DOE PAGES for author "Borgstahl, Gloria E. O."
• Search DOE PAGES for ORCID "0000-0001-8070-0258"

Publication Date:

Sat Jan 01 00:00:00 EST 2022

Research Org.:

Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States). Spallation Neutron Source (SNS)

Sponsoring Org.:

National Aeronautics and Space Administration (NASA); National Cancer Institute (NCI); National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1836134

Alternate Identifier(s):

OSTI ID: 1846512

Grant/Contract Number:  

AC05-00OR22725; P30CA036727

Resource Type:

Published Article

Journal Name:

Acta Crystallographica. Section F, Structural Biology Communications

Additional Journal Information:

Journal Name: Acta Crystallographica. Section F, Structural Biology Communications Journal Volume: 78 Journal Issue: 1; Journal ID: ISSN 2053-230X

Publisher:

International Union of Crystallography (IUCr)

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; human manganese superoxide dismutase; neutron diffraction; large unit cell; cryotrapping; peroxide