Proton-coupled electron transfer (PCET) is central to small-molecule activation processes, the function of redox-driven proton pumps and radical initiation and transport processes in biology. By examining PCET networks in model and natural systems, we aim to develop a mechanistic framework with which to interpret these processes. In doing so, we will contribute to an understanding of the structure/function relations of a variety of enzymes and proteins. This proposal seeks to elaborate PCET on three fronts: (1) The mechanism of PCET will be determined by undertaking timeresolved laser measurements on assemblies formed from a photoexcitable porphyrin donor (D) and acceptor (A) juxtaposed by a proton transfer interface (---[H+]---). The assemblies are designed to possess independent optical and vibrational signatures for electron and proton transfer events, thus allowing the fate of the proton in response to the electron and vice versa to be monitored by transient spectroscopies under a variety of conditions. These data and accompanying theoretical analysis will comprise a powerfully predictive framework for future interpretations of enzyme catalysis. (2) PCET will be studied in biological systems with the same mechanistic rigor that we have achieved in the foregoing model systems. The role of PCET in amino acid radical initiation and transport will be explored with the 35 A electron/proton coupled pathway in E. coli ribonucleotide reductase (RNR). Radicals will be generated from photoactive peptides or from non-natural amino acid photosensitizers, thereby bypassing the normal radical generation process originating at the diiron metallocofactor. The competency of these photoinitiated radicals at turning over RNR under various conditions (e.g., radical position along the pathway, variable effector and substrate concentrations) will be established using biochemical probes; the kinetics of radical transport will be investigated by transient laser spectroscopy. The combination of these steady-state and time-resolved studies should provide the most complete picture to date of PCET in a natural system. (3) The involvement of PCET in biological small molecule activation will be quantified with emphasis on bond-making and bond-breaking processes involving oxygen and water. PCET reactions will be investigated for protoporphyrin IX model cofactors that confine the delivery of protons and electrons in a face-to-face arrangement to bound O-O bonds and assembled oxygen atoms derived from water. These studies will provide direct insight into the PCET processes that are the underpinning of photosynthesis and respiration.