Real-time capture of domain movements during copper amine oxidase catalysis by mix-and-inject serial crystallography.

Abstract

Protein dynamics play a crucial role in various physiological functions, including enzyme catalysis. To explore conformational changes during enzyme catalysis, we conduct mix-and-inject serial crystallography, an advanced technique to capture time-resolved protein structures in real time, using the microcrystals of bacterial copper amine oxidase containing a protein-derived quinone cofactor. Within 50 ms of mixing the microcrystals (<4 μm) with a preferred substrate (2-phenylethylamine) under anaerobic conditions (reductive half-reaction), we observe domain movements associated with substrate binding and formation of a metastable reaction intermediate, a product Schiff-base of the quinone cofactor. At 100-1000 ms after mixing, conformational transition from aminoresorcinol to the semiquinone radical forms of the reduced cofactor progresses gradually, likely depending on the replacement of the product aldehyde by the next-cycle amine substrate that triggers the cofactor conformational change. Overall, this study provides structural insight into enzyme catalysis accompanying the active-site conformational changes that are hardly scrutinized by studies in solution.