Use of photocaged molecular oxygen for time-resolved serial X-ray crystallography
Abstract
Photocages offer an attractive means of synchronously triggering enzyme-substrate driven reactions in biological systems in crystallo expanding the reach of light-driven catalysis. We describe the application of photocaged molecular oxygen to trigger molecular oxygen binding in crystals of myoglobin under anaerobic conditions and follow structural changes using both serial synchrotron and serial femtosecond X-ray crystallography. This is enabled through use of fixed targets under anaerobic conditions, utilising thin polymeric films with low molecular oxygen permeability and validated by serially collecting deoxy myoglobin structures and complementary in crystallo UV-Vis spectroscopy. Release of molecular oxygen from the photocage and subsequent binding of the gaseous ligand is structurally visualised in oxygen-bound structures of myoglobin at 5 and 10 ms and various laser parameters. We present a robust workflow for enabling anaerobic room-temperature data collection of oxygen-sensitive samples on fixed targets and report the successful photo-release of caged molecular oxygen for time-resolved serial crystallography.
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