Visualising heme-catalysed activation of oxygen in real time

An outstanding frontier challenge in structural biology is to determine time-resolved structures at atomic resolution directly from systems engaged in catalysis, at physiological temperature and pressure. We and others hypothesize that most enzyme microcrystals (~2x2x2 m3 and smaller) will equilibrate with substrate(s) in s - ms, which is many times faster than the ~60 ms average enzyme turnover time in solution. We are targeting metalloenzyme catalysed activation of O2; a cornerstone of biology. The PhD studentship will visualise, in real time, the bond making and bond breaking events involved in heme- catalysed O2 activation with time-resolved serial crystallography methods at Diamond and at X-ray free electron laser (XFEL) facilities. In pursuing this ambitious goal, we will use many biophysical techniques that include time-resolved serial femtosecond crystallography (tr-SFX) combined with time- resolved X-ray emission spectroscopy (tr-XES) strategies to investigate reaction dynamics and structural characterisation of short-lived intermediates - ultimately producing a molecular movie of catalysis. The spectroscopic and crystallographic data will support electronic and atomic models of various species throughout reaction cycles, including Fe(III)-O-O- and Fe(IV)=O reactive species. These new serial crystallography methods allow us unprecedented opportunities to probe short lived intermediates at Diamond I24 and VMXi beamlines and at XFELs. Aspects of this PhD project will be carried out at the University of Bristol (School of Chemistry), Diamond Light Source (XFEL Hub at Diamond), and XFEL facilities around the world likely to include the LCLS in the USA, SACLA in Japan, PAL-XFEL in Korea, SwissFEL in Switzerland, and/or the European XFEL in Germany.

The project will involve training and use of the following methods: enzyme kinetics, various spectroscopies (uv-visible, EPR, resonance Raman), X-ray crystallography, and protein expression. No extensive prior experience of these methods is required, merely an ability and enthusiasm to apply chemical/biophysical principles to the study of metalloproteins.