CORDIS - EU research results

Binuclear Non-Coupled Copper Enzymes in Neurobiology: An Integrated Computational/Spectroscopic Investigation

Final Report Summary - ITCSCEN (Binuclear Non-Coupled Copper Enzymes in Neurobiology: An Integrated Computational/Spectroscopic Investigation)

Peptidylglycine α-hydroxylating monooxygenase (PAM) and dopamine β-monooxygense (DβM) are binuclear non-coupled copper enzymes that catalyze the hydroxylation of substrates in crucial importance for the nervous system. PAM catalyzes the amidation of the α-terminal carboxyl group of peptide neurotransmitters.The amidated peptides bind more strongly to the receptors and have longer half-life time and therefore more than 50% of all peptide neurotransmitters require such modification. This process is catalyzed by an peptidylglycine α-amidating monooxygenase PAM localized in the trans- Golgi network and secretory granules. It catalyzes two reactions and contains two separate enzymatic domains: peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine amidating lyase (PAL; EC 4.3.2) that work in sequential way on a glycine-extended propeptide. PHM is the rate limiting reaction.
Catalysis in both PAM and DβM is initiated by Cu reduction and O2 activation. Two possible mechanisms of O2 activation were discussed: i) via two electron reduction of O2 and formation of CuM(II) hydroperoxide; ii) through one electron reduction of O2 and formation of CuM-superoxide. The recent investigation performed on model compounds applying electronic structural/spectroscopic methods by the outgoing host group of this application indicated that one electron reduction path is favored.
Even with the structural information and the investigations of model compounds the mechanism of the hydroxylation reactions catalyzed by PAM and DβM remains unknown. Understanding electronic structure of the oxy-intermediates of the enzymes and their contribution to the reaction mechanisms is fundamental for design of new enzyme inhibitors and effective applications of the enzymes in biotechnology.
The aim of the present project is to understand the mechanisms of O2 activation, hydrogen abstraction, H2O binding and direct hydroxylation reactions catalyzed by the binuclear non-coupled copper enzymes PAM and DβM. The crucial starting point is the mechanism of O2 activation and revealing the chemical nature of the O2 adduct as it represents the initial structure for the subsequent steps of the reaction mechanism. We will investigate the oxidation state, the spin state, the chemical character of the O2 complexes utilizing an ensemble of spectroscopic methods and will analyze their electronic structural features by computational methods. Consequently we will characterize the geometric and electronic structure of the reactant complexes, transition states, reaction intermediates and product complexes within the protein environment. The Cu centres can be very complex to be investigated solely by computer or spectroscopy methods due to the electronic effects of the binuclear Cu-centres and the size and flexibility of enzyme molecule. Therefore we need to apply an integrated combination of the state-of-the art Computational Chemistry and Spectroscopy methods. The Computational Methods include Quantum Mechanical (QM), Molecular Dynamics (MD) and Combined Quantum Mechanical and Molecular Mechanical (QM/MM) Methods. Spectroscopic methods include X-ray absorption spectroscopy (XAS) combined with Circular Dichroism (CD), Magnetic Circular Dichroism (MCD), Variable-Temperature and Electron Paramagnetic Resonance (EPR).
In order to reach this aim we carried out an investigation of the electronic structure of the of the initial enzymatic reaction complex, including determination the oxidation state of Cu ion, defining the spin state of the complex, identifying the chemical nature of the O2 adduct (peroxide versus superoxide) using variety of spectroscopic methods and analysis of the chemical bonding and frontier molecular orbitals with QM methods. We calibrated and validated the theoretical methods in respect to the spectral data for the ground and excited states and modified of the density functionals until getting good agreement with measured spectra. Consequently we carried out an investigation of the reaction mechanism steps: H-abstraction, H2O binding and OH transfer, defining the reaction coordinate for each step, exploring the reaction paths, refining the transition states and possible reaction intermediates of each step. We also extended the study exploring of the conformational effects on the reaction mechanism using QM/MM and MD simulations. The spectroscopic properties of the catalytically important mutant forms have been revealed using multiple spectroscopic methods. The effects of multiple mutations on the active site residues of PHM have been modelled and explained and the conformational and dynamical properties of PAM and DβM have been explored and conformational effects of the reaction path of PHM have been analyzed. The gained results deepen and enhance our understanding about crucial steps of the enzyme mechanism of non-coupled di-copper containing enzymes and provide original perspectives for advanced applications in drug design, protein engineering and biotechnology.