Arizona State University

The use of serial femtosecond x-ray crystallography (SFX) is a recently developed technique in which a stream of nano - microcrystals are subjugated to an extremely brilliant femtosecond pulse of x-rays, generated by a free electron laser (FEL), that allow diffraction before the ensuing Coulomb explosion destroys the crystal ( Fig. 1 ). 4 The use of unique crystals for each diffraction pattern allows time - resolved SFX (TR - SFX) of fast and irreversible processes such as intermediate states and substrate dissociation . This can be achieved for photoactive proteins by introducing an optical photoexcitation to the protein stream at a specified time, τ, prior to interaction with the FEL, resulting in a populated transition state b eing probed. The technique allows the intermediate states of important biological reactions, such as the electron transport and subsequent undocking of ferredoxin (Fd) from photosystem I (PSI) and the transcis isomerization of retinol in rhodopsin, to be studied.3

PSI is a membrane protein complex that plays an integral role in photosynthesis, catalyzing the terminal step of photo - excited electron transport through the thylakoid membrane. This process results in the PSI bound protein Fd becoming reduced and undocking to participate in the reduction of NADP + to NADPH, which is crucial in the subsequent conversion of CO₂ into carbohydrates . The exact molecular mechanism by which Fd is reduced and successively undocks from PSI is unknown, although spectroscopic studies and preliminary SFX work have providd evidence that it is a multistep process with transition states that occur on the orders of nano and microseconds. 1,2 Revealing the electron transfer mechanism between PSI and Fd will be a significant step toward understanding of the full photosynthetic mechanism as well as improving our grasp of electron transfer within large membrane complexes.

Rhodopsin is a G - protein - coupled receptor that provides the basis for vision in many animals. Rhodopsin is activated through photoexcitation that leads to an irreversible reaction in which the cofactor retinal, bound through a Schiff base linkage, undergoes isomerization from 11 - cis - retinal to a ll - trans - retinal. This process proceeds through multiple intermediates, resulting in metarhodopsin II in which the Schiff base is deprotonated and is no longer covalently bound. The metarhodopsin II intermediate is the active state of rhodopsin that give s rise to the ensuing cascade chemical reaction that triggers the visual process. The aim of this research is to uncover the mechanism of electro n transfer between PSI and Fd and to establish undamaged structures of intermediates in the photolysis of rhodopsin, particularly metarhodopsin II.