Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1state and after double laser excitation (putative S3state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOxcubane in the S2to S3transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.