Structural insights into O2 activation and dynamics during Luciferase catalysis with a new fixed target device enabling rapid O2 reaction initiation

To understand how enzymes bind and activate O2 is of both basic scientific and, given the important roles of oxygenases in human biology, medicinal interest. Advances in X-ray sources have opened the door to room temperature investigation of reactions in microcrystals, i.e. time-resolved (tr) serial crystallography (SX). However, the current sample delivery systems available at synchrotron/XFEL endstations (fixed target chips, mixing jets, or drop on demand methods) are not suited for homogeneous and rapid initiation of in crystallo reactions by mixing with gases, including O2. I propose to develop an innovative fixed-target device that enables initiation of reactions with O2 for individual chip-mounted crystals in which a key element is in situ O2 generation. The bioluminescent luciferases that convert the chemical energy of O2 into light will be used as models to validate application of the new methods for tr-SX studies. The results will inform on how luciferases and related enzymes react with O2 and on dynamic structural changes and the nature of intermediates during luciferase catalysis. The pioneering new methodologies will lay the ground for a new generation of mixing possibilities in fixed-target devices and will be of general use in tr-S(S/F)X studies on O2-utilising enzymes and far beyond.