Phototriggers for time resolved crystallography in Heme enzymes

Time-resolved crystallography is a powerful technique for the study of enzyme dynamics, enabling visualization of intermediate state structures that are not easily inferred from static models of the reactant or product complexes. As it can be technically challenging to generate and freeze-trap intermediates in crystallo due to rapid reaction kinetics and the sensitive nature of protein crystals, the development of novel reaction initiation methods is highly desirable. Toward that end, we are exploring approaches to photosensitize heme-dependent enzymes by incorporating photoreductants and/or caged substrates that will enable in crystallo reaction initiation with light. We tested the reduction efficiencies of various photoreductants for a range of heme-dependent enzymes in solution and subsequently optimized settings for in crystallo reactions to achieve maximum efficiency with minimum crystal damage. We report the most success triggering photoreduction of the heme-dependent human immunosuppressive enzyme indoleamine 2,3-dioxygenase both in solution and in crystallo, along with promising results for initiating catalysis using a caged oxygen compound.