Learning from Nature hwo Two-metal Centres Operate: Studies of Purple Acid Phosphatases (PAP) and Polyphenol Oxidases (PPO)-Enzymes and Models

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The main objective of this project is to address the question of the respective roles of the two metal ions of two classes of bimetallic enzymes: the purple acid phosphatases (PAP) and the polyphenol oxidases (PPO with catechol oxidases (CO) and tyrosinase (Tyr)) via combination of structural and functional studies on enzymes and on model complexes. As a starting point, we will further advance the understanding of structurally-known systems (plant PAP and CO). Some controversies persist in the description of the mechanism of action at the molecular level and several aspects remain to be elucidated. A great deal of data are missing on the active species, the binding mode of the substrate (respective role of each metallic centre) and the importance and role of H+ and their transfer in the catalytic processes (pH dependence). In addition, we will focus our attention on highly important enzymes of the same family which to date remain poorly characterized: human PAP and Tyr. Based on our groups expertises, we will purify and attempt to obtain X-ray structures of these enzymes to assess the differences with the known structures of parent enzymes and get information through an overall comparison. Understanding molecular aspects of enzyme mechanisms is currently among the most challenging objectives in bioinorganic chemistry and using a synergistic interdisciplinary approach appears to be the most promising way of reaching this goal. The present project is built on such an approach and will include: 1. Isolation and purification of enzymes (High amount for well-known procedure - plant PAP and CO- and finalization of purification procedure - human PAP and Tyr) 2. Structural (X-ray) and biochemical studies (inhibitors, kinetics) 3. Biomimetic models to get reliable structural, spectroscopic and reactivity characteristics 4. Physical methods to investigate the molecular, electronic and magnetic structures of metal centres (models and enzymes)