Halsted, Thomas P 
ORCID: 0000-0002-5977-6511, Yamashita, Keitaro, Gopalasingam, Chai C, Shenoy, Rajesh T, Hirata, Kunio, Ago, Hideo, Ueno, Go, Blakeley, Matthew P, Eady, Robert R, Antonyuk, Svetlana V ORCID: 0000-0002-2779-9946 et al (show 2 more authors)
(2019)
Catalytically important damage-free structures of a copper nitrite reductase obtained by femtosecond X-ray laser and room-temperature neutron crystallography.
IUCRJ, 6 (Pt 4).
pp. 761-772.
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Abstract
Copper-containing nitrite reductases (CuNiRs) that convert NO<sub>2</sub> <sup>-</sup> to NO via a Cu<sub>CAT</sub>-His-Cys-Cu<sub>ET</sub> proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that Asp<sub>CAT</sub> and His<sub>CAT</sub> are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D<sub>2</sub>O) is positioned with one deuteron directed towards Asp<sub>CAT</sub> O<sup>δ1</sup> and one towards His<sub>CAT</sub> N<sup>∊2</sup>. The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D<sub>2</sub>O molecule as opposed to D<sub>3</sub>O<sup>+</sup> or OD<sup>-</sup>, which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated Asp<sub>CAT</sub> and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with Asp<sub>CAT</sub>. Upon the binding of NO<sub>2</sub> <sup>-</sup> a proton is transferred from the bridging water to the O<sup>δ2</sup> atom of Asp<sub>CAT</sub>, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from Asp<sub>CAT</sub> to the bound nitrite, enabling the reaction to proceed.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | copper-containing nitrite reductases, neutron crystallography, X-ray free-electron lasers |
| Depositing User: | Symplectic Admin |
| Date Deposited: | 08 Jul 2019 08:11 |
| Last Modified: | 19 Jan 2023 00:38 |
| DOI: | 10.1107/S2052252519008285 |
| Related URLs: | |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3048916 |
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