MS03-P06 Unreduced enzyme intermediate structure caught by X-ray Free Electron Laser Peter Moody (Leicester Institute for Structural & Chemical Biology, University of Leicester, Leicester, United Kingdom) Hanna Kwon (Leicester Institute for Structural & Chemical Biology, University of Leicester, Leicester, United Kingdom) Emma Raven ( Leicester Institute for Structural & Chemical Biology, University of Leicester, Leicester, United Kingdom) Takehiko Tosha (RIKEN SPring-8 Center, , Japan) Hiroshi Sugimoto (RIKEN SPring-8 Center, , Japan) Keitaro Yamashita (University of Tokyo, , Japan)email: peter.moody@le.ac.ukThe heme peroxidases have high valent ferryl (Fe(IV)) intermediates, these intermediates can be spectroscopically monitored, formed and cryo-trapped in the crystal, and thus the structures determined. However, X-rays are strongly reducing and therefore standard X-ray crystallographic data collection methods are likely to perturb the chemical nature of these intermediates. We are particularly interested in the “Compound II” intermediate, and determining if its identity is Fe(IV)=O or Fe(IV)-OH. These should be distinguishable from the Fe-O distance, but direct or indirect photo reduction into the ferric state would make these measurements invalid. By using fs flashes of X-rays from the free electron laser SACLA at Spring-8 to record diffraction data before photoreduction can take place, we have been able to determine the structure of the unreduced Compound II intermediate of Ascorbate Peroxidase at 1.5Å. The data collection methodology will be presented. The preliminary refinement results will be discussed in the context of our results from neutron crystallography and previous multiple crystal approaches.References:

Keywords: