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3-year project funded by Fondazione Cariparo (October 2024 - September 2027)
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Complete description of cofactor-free biological oxidative catalysis at atomic resolution
using state-of- the-art serial X-ray and neutron crystallography (SERIO2UOX)
In this project, using an integrated experimental and theoretical approach, we will address fundamental questions in O2-dependent biochemistry with an emphasis on the use of serial X-ray crystallographic approaches for the visualization of reaction intermediates, one of the most challenging tasks in structural enzymology. We will also employ neutron crystallography for the elucidation of critical protonation states. Our laboratory has a strong track record in structural biology and, amongst others, in the study of O2-dependent enzymes with important contributions in the field. Here, we will concentrate our attention on the archetypal cofactor-independent urate oxidase (UOX, EC 1.7.3.3).
The Great Oxidation Event around 2.4 billion years ago resulted in the accumulation of molecular oxygen (O2) in the atmosphere, profoundly shaping life as we know it today. For aerobes, O2 has played a pivotal role in metabolic reactions, driving the evolution of oxygenase and oxidase enzymes to harness its oxidative potential. Due to its stable triplet electronic ground-state, O2 requires activation to react. However, once activated, O2 can react indiscriminately, leading to detrimental consequences such as the formation of reactive oxygen species (ROS) implicated in aging. Thus, alongside activation, another challenge in oxygen biochemistry, is its control. The mechanisms by which triplet-state O2 reacts with singlet-state organic molecules, bypassing the quantum chemical spin-restriction rule, are fundamental questions in biochemistry. This is especially true for oxygenases and oxidases lacking redox transition metals or organic cofactors to facilitate this task. These cofactor-independent enzymes rely on limited chemical tools to activate O2, making them intriguing subjects for studying fundamental oxygen biochemistry and serving as model systems.In this project, we will investigate urate oxidase (UOX), the prototypical cofactor-independent oxidase responsible for the metabolism of uric acid. During evolution, UOX has disappeared in humans. For this reason, it is administered therapeutically in conditions leading to abnormal uric acid levels. Taking advantage of the ideal suitability of this experimental system we will provide a complete description of its reaction mechanism by integrating cutting-edge methodologies in structural biology (serial X-ray, serial electron, and neutron crystallography) with biochemical and theoretical approaches. Our overarching goal is to establish a general mechanistic framework for cofactor-independent O2 catalysis in biological systems.
