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

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.

Mononuclear Fe(II) metalloenzymes use the 2-His-1-carboxylate facial triad motif to activate molecular oxygen and produce highly specialised reagents able to carry out diverse biosynthetic reactions. 2,3-homoprotocatechuate dioxygenase (2,3-HPCD) from Brevibacterium fuscum is an extradiol dioxygenase that catalyses oxidative ring-opening of catecholic substrates, and is the model system for mononuclear Fe(II) metalloenzymes from the 2-His-1-carboxylate facial triad family. Recent work by Kovaleva and Lipscomb has demonstrated several advantages in probing reaction chemistry and protein dynamics of 2,3-HPCD in crystallo. The key intermediates in the proposed oxygen activation and extradiol ring-cleavage reaction were stabilised and characterised. These included substrate-semiquinone-Fe(II)-superoxo, Fe(II)-alkylperoxo and gem-diol intermediates, and represented the first structural insight into the mechanism of this enzyme class. In addition, the fact that under influence of crystal packing interactions nominally identical subunits can stabilise different species in the catalytic cycle strongly implies the involvement of protein dynamics in 2,3-HPCD catalysis. In this proposal we seek to gain further insight into the chemical and molecular mechanism of oxygen activation and extradiol ring cleavage reaction of 2,3-HPCD using X-ray and neutron crystallography, single crystal UV/vis and Raman spectroscopy and in silico methods. Specifically, we aim to 1) examine the influences that the electronic structure of substrates and acid-base and H-bonding properties of the active site have on intermediate stability in crystallo, 2) determine positions of catalytically relevant protons in the active site during various stages of catalysis, and 3) investigate the roles of protein dynamics and specific interactions within the active site in substrate and oxygen activation, insertion and ring-cleavage reactions.