Reaction intermediates in the O2 activation mechanism of an extradiol dioxygenase: visualising catalytic protons and active site dynamics in crystallo

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Oxygen is fundamental to life on earth, yet it is a surprisingly unreactive molecule that must be activated before it can be used by cells. Understanding how oxygen is activated is a critical question for life science researchers. Oxygen activation is mysterious and must be properly controlled for good health in humans. If the process happens uncontrollably, reactive oxygen species can be released into cells, causing damage linked to heart disease and ageing. In this project we are studying how an enzyme, a biological molecule that accelerates chemical reactions in the cell, activates oxygen. Our enzyme uses the activated oxygen to breakdown a family of chemicals that includes several natural and man-made pollutants. Understanding how this enzyme works will not only help explain how oxygen activation is controlled, but may also help in developing 'green chemistry' to clean up pollution. We intend examine this enzyme by bombarding it with intense X-ray light or a neutron beam in a technique known as crystallography. This method allows us to see events on the atomic level, so we can directly visualise how the oxygen interacts with the active centre of the enzyme. Excitingly, cooling the enzyme to -180C arrests the chemical reaction so that we can visualise the reaction at different stages. Combining crystallography with other techniques, like mutagenesis, where we alter the enzyme active centre, we can begin to understand the underlying chemical and physical processes that control oxygen activation and the ensuing chemistry. We will also employ computer modelling to discern chemical features we cannot currently observe in the lab. These computer models will first be validated against our known experimental data to ensure the results match. Then, they can be used to predict the results of experiments that are very difficult or impossible to do in real-life. We hope that by using this exciting combination of cutting-edge techniques we can achieve a more complete understanding of how chemistry involving oxygen activation is controlled by biology.