The aim of this proposal is to develop new photoactive tools able to isolate and trigger a specific catalytic event upon irradiation with a laser pulse. The laser pulse occurs at zero time, allowing for synchronization of initiation of catalysis, which can be monitored in a time-resolved manner. To date, caged compounds in which the active moiety is released from the caging group following laser excitation were primarily used to trigger reactions. The subsequent diffusion of the active moiety to the protein may be too slow and unsuited to time-resolved studies of proteins with fast turnover. This proposal represents a new approach to synchronize an ensemble of enzymes in solution, using a probe directly bound to the protein with the ability to trigger catalysis by ultrafast electrons injection to the protein. Overview: This project focuses on the use of novel photoactivable NADPH analogues, called nanotriggers (NTs), targeted to the NADPH site of nitric oxide synthase (NOS) isoforms and cytochrome P450 reductase (POR), to stabilize the enzymes in a closed conformation, facilitating crystallization. This project will: (1) determine the X-ray structure of various NT-protein complexes; (2) elucidate time-resolved structure/function studies of the first catalytic steps by kinetics studies in solution and by time-resolved X-ray crystallography; (3) design by in silico simulations and synthesis of novel isoform-specific eNOS activators; and (4) monitor eNOS trafficking in cells by biphotonic excitation, due to the intrinsic imaging properties and specificity of the NT probes. Broader Impacts: The photoactivation process of NT in which light absorption modulates the redox potential of the activated molecule (i.e., NT bound to NOS) and mediates electron flow is inspired from nature (i.e., photosynthesis), allowing the opening of new perspectives, especially in the field of artificial photosynthesis, a possible source of clean energy. In addition, the ability to trigger the activity of specific enzymes, particularly activation of eNOS, attenuated activity of which is a hallmark of endothelial dysfunction, open new therapeutic avenues for treatment of human diseases such as hypertension, diabetes, and degenerative neurological processes. Value of International Collaboration: The strength of this proposal lies in the novel collaborative multidisciplinary skills gathered in this consortium that combines all the expertise required for its realization through the complementarity of the French partners: J. Xie, chemical synthesis, A. Slama-Schwok, drug design and biophysics, E. Deprez, imaging and time-resolved fluorescence, and the American partners: L. Roman and J.J. Kim, biochemistry and structure of NOS and P450, respectively. Drs. Roman and Kim are long-time collaborators in their studies of NOS, and their respective expertise in NOS enzymology, protein chemistry, and molecular biology and X-ray crystallography approaches together with the French groups, chemical, synthetic, spectroscopic and computational expertise will yield extremely high synergy.