Capturing structural changes of the S₁ to S₂ transition of photosystem II using time-resolved serial femtosecond crystallography

Abstract

Photosystem II (PSII) catalyzes light-induced water oxidation through an S i -state cycle, leading to the generation of di-oxygen, protons and electrons. Pump–probe time-resolved serial femtosecond crystallography (TR-SFX) has been used to capture structural dynamics of light-sensitive proteins. In this approach, it is crucial to avoid light contamination in the samples when analyzing a particular reaction intermediate. Here, a method for determining a condition that avoids light contamination of the PSII microcrystals while minimizing sample consumption in TR-SFX is described. By swapping the pump and probe pulses with a very short delay between them, the structural changes that occur during the S1-to-S2 transition were examined and a boundary of the excitation region was accurately determined. With the sample flow rate and concomitant illumination conditions determined, the S2-state structure of PSII could be analyzed at room temperature, revealing the structural changes that occur during the S1-to-S2 transition at ambient temperature. Though the structure of the manganese cluster was similar to previous studies, the behaviors of the water molecules in the two channels (O1 and O4 channels) were found to be different. By comparing with the previous studies performed at low temperature or with a different delay time, the possible channels for water inlet and structural changes important for the water-splitting reaction were revealed.

The electron-proton bottleneck of photosynthetic oxygen evolution

“…The crystallographic analysis of semi-stable intermediates in the water oxidation cycle also supports reaction steps coupled to rearrangement of water molecules. 22,28 Our MEP calculations suggest protonation of the Asp61 carboxylate group in an electron transfer step that is coupled to a Grotthus-type movement of three protons, with the unconventional feature of a metal ion (Mn4) acting as a relay. This single-electron-multiproton step facilitates the critical step of Mn IV -O • formation (S4') prior to O-O bond formation.…”

The exchange of the fast substrate water in the S2 state of photosystem II is limited by diffusion of bulk water through channels – implications for the water oxidation mechanism

“…17,21,60,62,63 Recent room temperature and cryogenic X-ray crystallography studies favor that water access to the catalytic site occurs via the O1 channel as it shows the largest variation in water positions between studies and S states. 15,17,64 By contrast, previous theoretical studies suggested that water is delivered through the O4 channel to the Mn4 site and is inserted during the S 2 / S 3 transition via the pivot/carousel mechanism (Scheme 1C). 36,37 Recent mass spectrometric studies analyzing the oxidative damage to the D1, D2 and CP47 proteins caused by the formation of reactive oxygen species (ROS) at the Mn 4 CaO 5/6 cluster under illumination support both the B branch of the Cl1 channel and the O1 channel as water access pathways.…”

Potential of Time-Resolved Serial Femtosecond Crystallography Using High Repetition Rate XFEL Sources

“…In 2012, Aquila et al conducted the first TR-SFX experiments using the CFEL-ASG multi-purpose instrument at the Linac Coherent Light Source (LCLS) on the photosystem I-ferredoxin complex, where they used an Nd:YLF optical pump laser system to illuminate the complex and then probed the photo-induced structural changes that occurred within 5 to 10 µs delay times using the X-ray free electron laser [23]. This pioneering pump-probe TR-SFX experiment, accompanied by the advances in the application of pump lasers and microcrystal delivery methods, has paved the way to rapid development within the study of the structure and dynamics of light-sensitive biomacromolecules [18,24,[26][27][28]58]. In the pump-probe TR-SFX, an optical 'pump' laser is used to trigger the biochemical reaction in crystallo by illuminating the microcrystals with defined optical laser pulses at a defined wavelength and energy in a controlled region of the jet and/or nozzle before probing the reactions in crystallo with X-ray FEL pulses 'probe' at different delay times to allow observing the reaction of the biomacromolecule in real time (Figure 2) [18,23,24].…”

“…The selection of the optical pump setup, e.g., the laser wavelength, fluence and pulse duration, requires careful experimental planning to achieve as high as possible excited state population while avoiding contamination, which may occur, for instance, due to the multiphoton excitation of the chromophores [15,58,61,63]. Such multiphoton excitation can lead to the propagation of radical intermediates, which obscures the biological interpretation of the TR-SFX data [63,64].…”

“…Efficient photoexcitation protocol guided by the protein photochemical properties should be followed to allow for the biologically meaningful single-photon absorption of the biomolecules in crystals to avoid such an effect, i.e., multiphoton excitation. Moreover, examining the samples in their crystalline and solution forms with a static and time-resolved optical spectroscopy can significantly assist in the designing of the pump-probe TR-SFX experiments [28,58,61]. Such spectroscopic scanning, e.g., time-resolved UV-visible (transient) spectroscopy, can provide essential information that could help to establish the reaction photocycle, especially when examining a novel system [65], and estimate the time range of each of the photo-intermediates and highlight the differences, if any, between the crystalline and solution forms.…”