Serial crystallography captures dynamic control of sequential electron
and proton transfer events in a flavoenzyme
Nature Chemistry. 2022, April 7, DOI : 10.1038/s41557-022-00922-3
In all chemical reactions, such as reduction or oxidation, reactants are transformed into products. The pathways by which this transition takes place, and the substances populating them, which are called reaction intermediates, are at the core of modern chemistry. However, because chemical reactions are very fast, in the order of nano- to milliseconds, reaction intermediates are extremely challenging to study, and therefore only very limited information was available on their nature. Conversely, knowledge of reaction intermediates is crucial, as most advances in chemical technology rely on catalysts designed to modulate reaction pathways via stabilization of specific intermediates.
For the first time an international team led by Dr. Ming-Daw Tsai, Distinguished Visiting Chair of the Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, was able to determine the 3D structure of reaction intermediates in the light-dependent reduction of a photolyase enzyme at atomic resolution. Here, the reactive core of photolyase, called flavin adenine dinucleotide (FAD), was known to transform upon illumination into the reaction product FADH· after one reaction cycle, or FADH- after two. By using time-resolved crystallography at the Japanese Sub-Angstrom Compact X-ray Free Electron Laser (SACLA), a team of over 35 researchers managed by Academia Sinica visiting scientists Drs. Manuel Maestre-Reyna and Yoshitaka Bessho were able to collect snapshots at different times ranging from 10 ns to 5 ms, resulting in a 3D movie describing these FAD transformations in detail. This breakthrough was published as an article in Nature Chemistry on April 7th.
The 3D movie shows that, after the reaction is started by light, FAD changes its flat geometry into a distorted one within nanoseconds, in a motion reminiscent of the fluttering wings of a butterfly. Meanwhile, nearby amino-acids from the enzyme stabilize and support these movements, allowing the FAD to settle into the first semi-stable intermediate, FAD·-, which is characterized by a strong twist. Given the strained geometry of FAD·-, unless the protein environment quickly stabilizes it by donating a proton and yielding FADH·, photolyase reverts back to its original FAD state within milliseconds. If, however, FADH· is produced, a second reaction cycle elicits renewed fluttering in the FAD, resulting in the final product, FADH-.
By minutely analyzing the nature and structure of the reaction intermediates in a chemical reaction over time, this research represents a very significant contribution to our understanding of fundamental chemistry. Furthermore, it is the first step in the road towards rational catalyst design based on their true binding partners, i.e. neither reactants nor products, but rather reaction intermediates.

(Click on the figure above to see a 3D movie on the FAD)
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