Horrell, Sam and Kekilli, Demet and Sen, Kakali and Owen, Robin U and Dworkowski, Florian SN and Antonyuk, Svetlana V and Keal, Thomas W and Yong, Chin W and Eady, Robert R and Hasnain, S Samar and Strange, Richard W and Hough, Michael A (2018) Enzyme catalysis captured using multiple structures from one crystal at varying temperatures. IUCrJ, 5 (3). pp. 283-292. DOI https://doi.org/10.1107/S205225251800386X
Horrell, Sam and Kekilli, Demet and Sen, Kakali and Owen, Robin U and Dworkowski, Florian SN and Antonyuk, Svetlana V and Keal, Thomas W and Yong, Chin W and Eady, Robert R and Hasnain, S Samar and Strange, Richard W and Hough, Michael A (2018) Enzyme catalysis captured using multiple structures from one crystal at varying temperatures. IUCrJ, 5 (3). pp. 283-292. DOI https://doi.org/10.1107/S205225251800386X
Horrell, Sam and Kekilli, Demet and Sen, Kakali and Owen, Robin U and Dworkowski, Florian SN and Antonyuk, Svetlana V and Keal, Thomas W and Yong, Chin W and Eady, Robert R and Hasnain, S Samar and Strange, Richard W and Hough, Michael A (2018) Enzyme catalysis captured using multiple structures from one crystal at varying temperatures. IUCrJ, 5 (3). pp. 283-292. DOI https://doi.org/10.1107/S205225251800386X
Abstract
High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.
Item Type: | Article |
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Uncontrolled Keywords: | serial crystallography; copper nitrite reductase; variable temperature; radiolysis; structural dynamics; density functional theory |
Subjects: | Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology |
Divisions: | Faculty of Science and Health Faculty of Science and Health > Life Sciences, School of |
SWORD Depositor: | Unnamed user with email elements@essex.ac.uk |
Depositing User: | Unnamed user with email elements@essex.ac.uk |
Date Deposited: | 15 May 2018 13:59 |
Last Modified: | 30 Oct 2024 16:00 |
URI: | http://repository.essex.ac.uk/id/eprint/22019 |
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