Yoshiki Nakajima

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

Photosystem II (PSII) is a huge membrane-protein complex consisting of 20 different subunits with a total molecular mass of 350 kDa for a monomer. It catalyses light-driven water oxidation at its catalytic centre, the oxygen-evolving complex (OEC). The structure of PSII has been analysed at 1.9 Å resolution by synchrotron radiation X-rays, which revealed that the OEC is a Mn4CaO5 cluster organized in an asymmetric, ‘distorted-chair’ form. This structure was further analysed with femtosecond X-ray free electron lasers (XFEL), providing the ‘radiation damage-free’ structure. The mechanism of O=O bond formation, however, remains obscure owing to the lack of intermediate-state structures. Here we describe the structural changes in PSII induced by two-flash illumination at room temperature at a resolution of 2.35 Å using time-resolved serial femtosecond crystallography with an XFEL provided by the SPring-8 ångström compact free-electron laser. An isomorphous difference Fourier map between the two-flash and dark-adapted states revealed two areas of apparent changes: around the QB/non-haem iron and the Mn4CaO5 cluster. The changes around the QB/non-haem iron region reflected the electron and proton transfers induced by the two-flash illumination. In the region around the OEC, a water molecule located 3.5 Å from the Mn4CaO5 cluster disappeared from the map upon two-flash illumination. This reduced the distance between another water molecule and the oxygen atom O4, suggesting that proton transfer also occurred. Importantly, the two-flash-minus-dark isomorphous difference Fourier map showed an apparent positive peak around O5, a unique μ4-oxo-bridge located in the quasi-centre of Mn1 and Mn4 (refs 4,5). This suggests the 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 consistent with that proposed previously.

Optical identification of the long-wavelength (700-1700 nm) electronic excitations of the native reaction centre, Mn4CaO5 cluster and cytochromes of photosystem II in plants and cyanobacteria

Visible/UV absorption in PS II core complexes is dominated by the chl-a absorptions, which extend to ~700 nm. A broad 700-730 nm PS II core complex absorption in spinach has been assigned to a charge transfer excitation between ChlD1 and ChlD2. Emission from this state, which peaks at 780 nm, has been seen for both plant and cyanobacterial samples. We show that Thermosynechococcus vulcanus PS II core complexes have parallel absorbance in the 700-730 nm region and similar photochemical behaviour to that seen in spinach. This establishes the low energy charge transfer state as intrinsic to the native PS II reaction centre. High-sensitivity MCD measurements made in the 700-1700 nm region reveal additional electronic excitations at ~770 nm and ~1550 nm. The temperature and field dependence of MCD spectra establish that the system peaking near 1550 nm is a heme-to-Fe(III) charge transfer excitation. These transitions have not previously been observed for cyt b559 or cyt c550. The distinctive characteristics of the MCD signals seen at 770 nm allow us to assign absorption in this region to a dz(2)→d(x2-y2) transition of Mn(III) in the Ca-Mn4O5 cluster of the oxygen evolving centre. Current measurements were performed in the S1 state. Detailed analyses of this spectral region, especially in higher S states, promise to provide a new window on models of water oxidation.

Proton Matrix ENDOR Studies on Ca2+-depleted and Sr2+-substituted Manganese Cluster in Photosystem II.

Background: Ca2+ plays an essential role in oxygen evolution in photosystem II. Results: ENDOR spectra showed that some protons surrounding the manganese cluster are lost and modified by Ca2+ depletion. Conclusion: The proton signals arising from water molecules ligated to the Ca2+ were identified. Significance: The roles of Ca2+ are the maintenance of the hydrogen bond network near the Ca2+ site and electron transfer pathway to the manganese cluster. Proton matrix ENDOR spectra were measured for Ca2+-depleted and Sr2+-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca2+-containing and Sr2+-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca2+-containing and Sr2+-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca2+-containing and Sr2+-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca2+-containing and Sr2+-substituted PSII because of modifications of the water protons ligating to the Sr2+ ion. Importantly, Ca2+ depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca2+ and YZ directly. With respect to the crystal structure and the functions of Ca2+ in oxygen evolution, it was concluded that the roles of Ca2+ and Sr2+ involve the maintenance of the hydrogen bond network near the Ca2+ site and electron transfer pathway to the manganese cluster.

Structured near-infrared Magnetic Circular Dichroism spectra of the Mn4CaO5 cluster of PSII in T. vulcanus are dominated by Mn(IV) d-d ‘spin-flip’ transitions

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn4CaO5. VTVH MCD spectra taken in the S2 state provide a clear g=2, S=1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749nm, negative (-) at 773nm and (+) at 808nm are assigned as 4A→2E spin-flips within the d3 configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S3 exhibits a more intense (-) MCD peak at 764nm and has a stronger MCD saturation characteristic. This S3 MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S2 EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.

Carcinoma infection and immune systems of Ehrlich ascites carcinoma-bearing mice treated with structurally similar sulfonium compounds.

The effects of intraperitoneal administration of dimethylsulfonioacetate (DMSA), dimethlsulfoniopropionate (DMSP), and methylmethionine (MeMet) solutions (10 mM each) on the body weights and the hematological parameters (red and white blood cells) of Ehrlich ascites carcinoma (EAC)-bearing mice were examined for up to 10 d. Body weights significantly increased in the EAC-bearing mice treated with and without MeMet in contrast to those with DMSA and DMSP. This increase was attributed to the increased amounts of ascitic fluid. EAC-bearing mice with and without MeMet both showed abnormal values of hematological parameters, while those with DMSA and DMSP exhibited almost normal levels on the 10th day.

Fourier Transform Infrared Analysis of the S-State Cycle of Water Oxidation in the Microcrystals of Photosystem II

Photosynthetic water oxidation is performed in photosystem II (PSII) through a light-driven cycle of intermediates called S states (S0-S4) at the water oxidizing center. Time-resolved serial femtosecond crystallography (SFX) has recently been applied to the microcrystals of PSII to obtain the structural information on these intermediates. However, it remains unanswered whether the reactions efficiently proceed throughout the S-state cycle retaining the native structures of the intermediates in PSII crystals. We investigated the water oxidation reactions in the PSII microcrystals using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. In comparison with the FTIR spectra in solution, it was shown that all of the metastable intermediates in the microcrystals retained their native structures, and the efficiencies of the S-state transitions remained relatively high, although those of the S2 → S3 and S3 → S0 transitions were slightly lowered possibly due to some restriction of water movement in the crystals.

Thylakoid membrane lipid sulfoquinovosyl-diacylglycerol (SQDG) is required for full functioning of photosystem II in Thermosynechococcus elongatus

Sulfoquinovosyl-diacylglycerol (SQDG) is one of the four lipids present in the thylakoid membranes. Depletion of SQDG causes different degrees of effects on photosynthetic growth and activities in different organisms. Four SQDG molecules bind to each monomer of photosystem II (PSII), but their role in PSII function has not been characterized in detail, and no PSII structure without SQDG has been reported. We analyzed the activities of PSII from an SQDG-deficient mutant of the cyanobacterium Thermosynechococcus elongatus by various spectroscopic methods, which showed that depletion of SQDG partially impaired the PSII activity by impairing secondary quinone (QB) exchange at the acceptor site. We further solved the crystal structure of the PSII dimer from the SQDG deletion mutant at 2.1 Å resolution and found that all of the four SQDG-binding sites were occupied by other lipids, most likely PG molecules. Replacement of SQDG at a site near the head of QB provides a possible explanation for the QB impairment. The replacement of two SQDGs located at the monomer–monomer interface by other lipids decreased the stability of the PSII dimer, resulting in an increase in the amount of PSII monomer in the mutant. The present results thus suggest that although SQDG binding in all of the PSII-binding sites is necessary to fully maintain the activity and stability of PSII, replacement of SQDG by other lipids can partially compensate for their functions.