Two Different Structures of the Oxygen-Evolving Complex in the Same Polypeptide Frameworks of Photosystem II

The oxygen-evolving complex (OEC) forms the heart of photosystem II (PSII) in photosynthesis. The crystal structure of PSII from Thermosynechococcus vulcanus has been reported at a resolution of 1.9 Å and at an averaged X-ray dose of 0.43 MGy. The OEC structure is suggested to be partially reduced to Mn­(II) by EXAFS and DFT computational studies. Recently, the “radiation-damage-free” structures have been published at 1.95 Å resolution using XFEL, but reports continued to appear that the OEC is reduced to the S₀-state of the Kok cycle. To elucidate much more precise structure of the OEC, in this study two structures were determined at extremely low X-ray doses of 0.03 and 0.12 MGy using conventional synchrotron radiation source. The results indicated that the X-ray reduction effects on the OEC were very small in the low dose region below 0.12 MGy, that is, a threshold existed for the OEC structural changes caused by X-ray exposure. The OEC structures of the two identical monomers in the crystal were clearly different under the threshold of the radiation dose, although the surrounding polypeptide frameworks of PSII were the same. The assumption that the OECs in the crystal were in the dark-stable S₁-state of the Kok cycle should be re-evaluated

ADP-Ribose Pyrophosphatase Reaction in Crystalline State Conducted by Consecutive Binding of Two Manganese(II) Ions as Cofactors

Adenosine diphosphate ribose pyrophosphatase (ADPRase), a member of the Nudix family proteins, catalyzes the metal-induced and concerted general acid–base hydrolysis of ADP ribose (ADPR) into AMP and ribose-5′-phosphate (R5P). The ADPR-hydrolysis reaction of ADPRase from <i>Thermus thermophilus</i> HB8 (<i>Tt</i>ADPRase) requires divalent metal cations such as Mn²⁺, Zn²⁺, or Mg²⁺ as cofactors. Here, we report the reaction pathway observed in the catalytic center of <i>Tt</i>ADPRase, based on cryo-trapping X-ray crystallography at atomic resolutions around 1.0 Å using Mn²⁺ as the reaction trigger, which was soaked into <i>Tt</i>ADPRase-ADPR binary complex crystals. Integrating 11 structures along the reaction timeline, five reaction states of <i>Tt</i>ADPRase were assigned, which were ADPRase alone (E), the ADPRase-ADPR binary complex (ES), two ADPRase-ADPR-Mn²⁺ reaction intermediates (ESM, ESMM), and the postreaction state (E′). Two Mn²⁺ ions were inserted consecutively into the catalytic center of the ES-state and ligated by Glu86 and Glu82, which are highly conserved among the Nudix family, in the ESM- and ESMM-states. The ADPR-hydrolysis reaction was characterized by electrostatic, proximity, and orientation effects, and by preferential binding for the transition state. A new reaction mechanism is proposed, which differs from previous ones suggested from structure analyses with nonhydrolyzable substrate analogues or point-mutated ADPRases

Deformation of Chlorin Rings in the Photosystem II Crystal Structure

The crystal structure of Photosystem II (PSII) analyzed at a resolution of 1.9 Å revealed deformations of chlorin rings in the chlorophylls for the first time. We investigated the degrees of chlorin ring deformation and factors that contributed to them in the PSII crystal structure, using a normal-coordinate structural decomposition procedure. The out-of-plane distortion of the P_D1 chlorin ring can be described predominantly by a large “doming mode” arising from the axial ligand, D1-His198, as well as the chlorophyll side chains and PSII protein environment. In contrast, the deformation of P_D2 was caused by a “saddling mode” arising from the D2-Trp191 ring and the doming mode arising from D2-His197. Large ruffling modes, which were reported to lower the redox potential in heme proteins, were observed in P_D1 and Chl_D1, but not in P_D2 and Chl_D2. Furthermore, as P_D1 possessed the largest doming mode among the reaction center chlorophylls, the corresponding bacteriochlorophyll P_L possessed the largest doming mode in bacterial photosynthetic reaction centers. However, the majority of the redox potential shift in the protein environment was determined by the electrostatic environment. The difference in the chlorin ring deformation appears to directly refer to the difference in “the local steric protein environment” rather than the redox potential value in PSII

Evidence for an Unprecedented Histidine Hydroxyl Modification on D2-His336 in Photosystem II of <i>Thermosynechoccocus vulcanus</i> and <i>Thermosynechoccocus elongatus</i>

The electron density map of the 3D crystal of Photosystem II from Thermosynechococcus vulcanus with a 1.9 Å resolution (PDB: 3ARC) exhibits, in the two monomers in the asymmetric unit cell, an, until now, unidentified and uninterpreted strong difference in electron density centered at a distance of around 1.5 Å from the nitrogen Nδ of the imidazole ring of D2-His336. By MALDI-TOF/MS upon tryptic digestion, it is shown that ∼20–30% of the fragments containing the D2-His336 residue of Photosystem II from both Thermosynechococcus vulcanus and Thermosynechococcus elongatus bear an extra mass of +16 Da. Such an extra mass likely corresponds to an unprecedented post-translational or chemical hydroxyl modification of histidine

Oxygen-Evolving Porous Glass Plates Containing the Photosynthetic Photosystem II Pigment–Protein Complex

The development of artificial photosynthesis has focused on the efficient coupling of reaction at photoanode and cathode, wherein the production of hydrogen (or energy carriers) is coupled to the electrons derived from water-splitting reactions. The natural photosystem II (PSII) complex splits water efficiently using light energy. The PSII complex is a large pigment–protein complex (20 nm in diameter) containing a manganese cluster. A new photoanodic device was constructed incorporating stable PSII purified from a cyanobacterium Thermosynechococcus vulcanus through immobilization within 20 or 50 nm nanopores contained in porous glass plates (PGPs). PSII in the nanopores retained its native structure and high photoinduced water splitting activity. The photocatalytic rate (turnover frequency) of PSII in PGP was enhanced 11-fold compared to that in solution, yielding a rate of 50–300 mol e^–/(mol PSII·s) with 2,6-dichloroindophenol (DCIP) as an electron acceptor. The PGP system realized high local concentrations of PSII and DCIP to enhance the collisional reactions in nanotubes with low disturbance of light penetration. The system allows direct visualization/determination of the reaction inside the nanotubes, which contributes to optimize the local reaction condition. The PSII/PGP device will substantively contribute to the construction of artificial photosynthesis using water as the ultimate electron source