MOLECULAR BASIS FOR THE DIFFERENTIAL REACTIVITY OF COPPER SITES IN PHM

DIEGO MORENO; PETER INSLEY; LEONARDO BOECHI; DARIO A. ESTRIN; DAMIAN A. SCHERLIS

Buzios-Brasil

Congreso; VII Congreso Iberoamericano de Biofìsica; 2009

Objectives The enzyme peptidylglycine-alpha-hydroxylating monooxygenase (PHM) binds molecular oxygen to catalyze the hydroxylation of the Cα of a C-terminal glycine. Two redox sites have been identified in PHM, consisting of two inequivalent Cu centers (CuH or CuA and CuM or CuB) widely separated in space (≈11 Å). In spite of crystallographic studies indicating that both copper centers have similar enviroments,1 dioxygen binds to CuB only, which is the site where hydroxylation occurs. In this work we seek to explain the reason for the difference in the oxygen affinities observed between both metallic sites. Methods We performed computational calculations in small model systems and in the full protein. The studies in the small active site models were carried out within the DFT framework with the Gaussian 03 package using the PBE xc-functional. QM-MM (Quantum Mechanics-Molecular Mechanics) calculations including the full protein plus solvation water molecules were realized using the SIESTA program, combining DFT to describe the active site and the Amber99 force field parameterization to represent the environment. Results Our simulations show no significant difference between the oxygen affinities of both copper sites. The trends observed in the small model systems and in the protein treated under the QM-MM approach are similar. On the other hand, classical MD simulations at 298 K suggest that the coordination to the metal center could be different from the one obtained in the crystallographic studies. Conclusions Our results suggest a change in the coordination of the CuA active site (CuA-His3 to CuA-His3L, L=ligand), which could be responsible for the saturation of the coordination sphere of copper, explaining its lack of reactivity. 1. Science, 304:864-867,2004 Methods We performed computational calculations in small model systems and in the full protein. The studies in the small active site models were carried out within the DFT framework with the Gaussian 03 package using the PBE xc-functional. QM-MM (Quantum Mechanics-Molecular Mechanics) calculations including the full protein plus solvation water molecules were realized using the SIESTA program, combining DFT to describe the active site and the Amber99 force field parameterization to represent the environment. Results Our simulations show no significant difference between the oxygen affinities of both copper sites. The trends observed in the small model systems and in the protein treated under the QM-MM approach are similar. On the other hand, classical MD simulations at 298 K suggest that the coordination to the metal center could be different from the one obtained in the crystallographic studies. Conclusions Our results suggest a change in the coordination of the CuA active site (CuA-His3 to CuA-His3L, L=ligand), which could be responsible for the saturation of the coordination sphere of copper, explaining its lack of reactivity. 1. Science, 304:864-867,2004 Å). In spite of crystallographic studies indicating that both copper centers have similar enviroments,1 dioxygen binds to CuB only, which is the site where hydroxylation occurs. In this work we seek to explain the reason for the difference in the oxygen affinities observed between both metallic sites. Methods We performed computational calculations in small model systems and in the full protein. The studies in the small active site models were carried out within the DFT framework with the Gaussian 03 package using the PBE xc-functional. QM-MM (Quantum Mechanics-Molecular Mechanics) calculations including the full protein plus solvation water molecules were realized using the SIESTA program, combining DFT to describe the active site and the Amber99 force field parameterization to represent the environment. Results Our simulations show no significant difference between the oxygen affinities of both copper sites. The trends observed in the small model systems and in the protein treated under the QM-MM approach are similar. On the other hand, classical MD simulations at 298 K suggest that the coordination to the metal center could be different from the one obtained in the crystallographic studies. Conclusions Our results suggest a change in the coordination of the CuA active site (CuA-His3 to CuA-His3L, L=ligand), which could be responsible for the saturation of the coordination sphere of copper, explaining its lack of reactivity. 1. Science, 304:864-867,2004 Methods We performed computational calculations in small model systems and in the full protein. The studies in the small active site models were carried out within the DFT framework with the Gaussian 03 package using the PBE xc-functional. QM-MM (Quantum Mechanics-Molecular Mechanics) calculations including the full protein plus solvation water molecules were realized using the SIESTA program, combining DFT to describe the active site and the Amber99 force field parameterization to represent the environment. Results Our simulations show no significant difference between the oxygen affinities of both copper sites. The trends observed in the small model systems and in the protein treated under the QM-MM approach are similar. On the other hand, classical MD simulations at 298 K suggest that the coordination to the metal center could be different from the one obtained in the crystallographic studies. Conclusions Our results suggest a change in the coordination of the CuA active site (CuA-His3 to CuA-His3L, L=ligand), which could be responsible for the saturation of the coordination sphere of copper, explaining its lack of reactivity. 1. Science, 304:864-867,2004