Time-resolved crystallography reveals allosteric communication aligned with molecular breathing.

A comprehensive understanding of protein function demands correlating structure and dynamic changes. Using time-resolved serial synchrotron crystallography, we visualized half-of-the-sites reactivity and correlated molecular-breathing motions in the enzyme fluoroacetate dehalogenase. Eighteen time points from 30 milliseconds to 30 seconds cover four turnover cycles of the irreversible reaction. They reveal sequential substrate binding, covalent-intermediate formation, setup of a hydrolytic water molecule, and product release. Small structural changes of the protein mold and variations in the number and placement of water molecules accompany the various chemical steps of catalysis. Triggered by enzyme-ligand interactions, these repetitive changes in the protein framework's dynamics and entropy constitute crucial components of the catalytic machinery.