Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL

Abstract

SummaryPhotosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer, and catalyzes light-driven water oxidation at its catalytic center, the oxygen-evolving complex (OEC) [1][2][3] . The structure of PSII has been analyzed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that OEC is a Mn4CaO5 cluster organized in an asymmetric, "distorted-chair" form 4 . This structure was further analyzed with femtosecond X-ray free electron lasers (XFEL), providing the "radiation damage-free" 5 structure. The mechanism of O=O bond formation, however, remains obscure due to the lack of intermediate state structures. Here we report the structural changes of PSII induced by 2-flash (2F) illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography (TR-SFX) with an XFEL provided by the SPring-8 angstrom compact free-electron laser (SACLA). Isomorphous differenceFourier map between the 2F and dark-adapted states revealed two areas of apparent changes; they are around QB/non-heme iron and the Mn4CaO5 cluster. The changes around the QB/non-heme iron region reflected the electron and proton transfers induced by the 2F-illumination. In the region around the Mn4CaO5 cluster, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon 2Fillumination, leading to a closer distance between another water molecule and O4, suggesting also the occurrence of proton transfer. Importantly, the 2F-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ3-oxo-bridge located in the quasi-center of Mn1 and Mn4 4,5 . This suggests an insertion of a new oxygen atom (O6) close to O5, providing an O=O distance of 1.5 Å between these two oxygen atoms. This provides a mechanism for the O=O bond formation 4 consistent with that proposed by Siegbahn 6,7 . Fig. 1a shows organization of the electron transfer chain of PSII in a pseudo-C2 symmetry by two subunits D1 and D2. The water-oxidation reaction proceeds via the Si-state cycle 8 (with i=0-4), where dioxygen is produced in the transition of S3→(S4)→S0 (Fig. 1b). The high-resolution structures of PSII analyzed so far were for the dark-stable S1 state 4,5 , although a few studies on the low-resolution intermediate S-state structures have been reported by TR-SFX [9][10][11] . During the revision of our manuscript, Young et al. reported a 2F-illuminated state structure at 2.25 Å resolution where no apparent changes around O5 were observed 12 , although estimations of the resolution could yield somewhat different values so that small movement of some water molecules may escape the detection. In order to achieve resolution high enough to uncover small structural changes induced by flash illuminations yet allowing Si-state transition to proceed efficiently, we determined the optimal crystal size of PSII with a maximum length of 100 µm, which diffracted up to a resolution of 2.1 Å by a SACLA-XFEL ...

Mechanistic Parameters of Electrocatalytic Water Oxidation on LiMn2O4 in Comparison to Natural Photosynthesis

“…However, O−H bond breaking should have fast kinetics for high concentrations of OH − (p K a  14) in alkaline electrolytes and thus dioxygen formation or release are most likely limiting. It should be noted that the details of the latter two steps are also still debated for natural photosynthesis 9, 10, 11, 12, 20, 21, 22, 23, 94, 95…”

“…The structure of the active site is known with high resolution8, 9, 19 and furthermore, the active states have been studied for decades by various experimental and theoretical methods 2, 10, 11, 12, 20, 21, 22, 23. The mechanism of water oxidation in natural photosynthesis is the so‐called S‐state cycle or Kok cycle24 (Figure 1).…”

“…Light flashes advance the catalytic cycle by oxidizing Mn ions to Mn 4+ in state S 3 . This high‐valent state drives oxygen evolution at the site marked with an asterisk after an additional light flash 9. The state resulting after oxygen evolution, S 0 , can then be oxidized back to state S 1 by a fourth flash.…”

Status of the SACLA Facility

“…Furthermore, they determined the structure of an intermediate S 3 state with two-flash illumination at room temperature at a resolution of 2.35 Å with a time-resolved (TR) serial femtosecond crystallography (SFX) method. This finding suggests the insertion of a new oxygen atom close to an existing oxygen atom in the molecule [11]. These results provide a critical basis for understanding the mechanisms underlying oxygen evolution in photosynthesis.…”

“…To enable this, we employed unique accelerator technologies: a low emittance injector with a thermionic electron gun (e-gun) and a velocity bunching system; a high-gradient normal-conducting C-band linac; and a short-period in-vacuum undulator [4]. Combining these devices with state-of-the-art X-ray optics [5,6], we have steadily generated XFEL light for users more than 4000 h in FY2016, allowing researchers to produce a number of important scientific results in the fields of biology [7][8][9][10][11][12], chemistry [13][14][15][16], materials science [17][18][19][20], high-energy density science [21], and non-linear X-ray optics [22][23][24][25][26]. The success of SACLA has promoted the development of similar compact XFEL facilities, such as SwissFEL at Paul Scherrer Institut in Switzerland [27].…”