The exact chemical identity of reactive intermediates in O2-dependent uric acid biodegradation

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Differently from the majority of other animals humans cope with large quantities of uric acid in their bodies. This is because during evolution we have progressively silenced a gene responsible for the production of an enzyme called urate oxidase (UOX). This enzyme is able to break down uric acid into more soluble compounds. The reasons for human adaptation to high uric acid levels are not entirely clear and, interestingly, mice without a functional UOX enzyme die shortly after birth. Under certain pathological conditions that cause a further increase in uric acid UOX is administered to patients to help restore normal levels. Crystalline uric acid deposits are also the hallmark of gout disease.UOX requires molecular oxygen (O2) to perform its task of breaking down uric acid. O2 is a very interesting molecule as in its normal "resting" state (the form present in the air) does not want to react with the vast majority of organic molecules for reasons related to its electronic structure. Oxygen needs activation to react. A major problem, however, is that once "activated" oxygen can react indiscriminately with many biological molecules with detrimental consequences. For example, reactive oxygen species (ROS) are damaging forms of "active oxygen" that play an important role in aging. Therefore, besides the generation of "active oxygen", another challenge in oxygen biochemistry, is its control. In this work we will investigate how UOX uses O2 to break down uric acid. Interestingly, UOX belongs to a small group of enzymes that can bring oxygen into reacting with their organic substrates and steer the reaction towards the desired products with limited chemical tools at its disposal. In fact, as oxygen activation is not an easy task, the vast majority of enzymes rely on special additional components like metal and/or organic co-factors to form "active oxygen". UOX does not require these additional helpers and therefore understanding how it works is particularly intriguing. Using a technique called X-ray crystallography which allows to 'see' at very high resolution the 3D structure of molecules as small as urate oxidase (10,000 times smaller that the thickness of a human's hair) we have been able to visualise snapshots of the enzyme along the process of uric acid degradation (reaction intermediates) including also the state in which O2 is trapped above the substrate. These snapshots led us to formulate some hypotheses on how urate oxidase works. We are now in an excellent position to study the most elusive and critically important properties of UOX chemistry. For this we will use a technique called neutron crystallography that can detect atoms (hydrogens) that cannot be typically observed even by X-ray crystallography. By combining neutron crystallography, X-ray crystallography, modern spectroscopic techniques and advanced quantum mechanical theoretical methods to probe states that are not experimentally accessible we will understand general rules of O2 biochemistry in the context of UOX function. This integrated approach will allow a deeper understanding not only of UOX but also of oxygen, an essential component of life on Earth.