Neutron crystallography of a heme peroxidase

Heme peroxidases are an ubiquitous family of catalytic iron-containing proteins. These enzymes are responsible for the catalysis of hydrogen peroxide removal from the cell through the formation of high valent transient states of the heme cofactor. The purpose of the present research project is to investigate the reaction pathway of cytochrome c peroxidase. The study of hydrogen related chemical features in the active site of this enzyme in the resting state and in the transient species is of paramount importance for the clarification of the reaction mechanism. In these circumstances neutron crystallography is the technique of choice. Neutron crystallography allows the direct localization of deuterium substituted hydrogen atoms in macromolecules in absence of radiation-induced damage. The structures of the resting state and of cryo-trapped compound I of cytochrome c peroxidase were determined by neutron crystallography. The nature of the catalytic center was investigated and in particular the protonation state of the heme iron axial ligand and of the key catalytic residues was established. These findings contributed to the understanding of the reaction pathway from the resting state to the intermediate species compound I. Complementary spectroscopic techniques were employed to assess compound I formation in single crystals and its stability in the conditions of the neutron crystallography experiment. The temperature dependence of the key catalytic features of cytochrome c peroxidase in the resting state was investigated by neutron crystallography. Significant alterations of protonation states were found in the resting state at cryogenic temperature. In addition the nature of the heme iron distal ligand was found to be affected by temperature in the resting state and it was possible to relate this phenomenon to the temperature induced spin state change observed by spectrophotometry in the visible region. As part of the present project, contribution was given to the development of the cryogenic temperature sample environment at the neutron macromolecular diffractometer LADI-III of the Institut Laue-Langevin broadening the experimental capabilities of the instrument. A wide range of experiments are made possible by the new set-up including the cryo-trapping of catalytic intermediates, the study of the temperature dependence of structural features and the investigation of species and complexes that are not stable at ambient temperature.