Keisuke Kawakami

Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 A

Photosystem II is the site of photosynthetic water oxidation and contains 20 subunits with a total molecular mass of 350 kDa. The structure of photosystem II has been reported at resolutions from 3.8 to 2.9 Å. These resolutions have provided much information on the arrangement of protein subunits and cofactors but are insufficient to reveal the detailed structure of the catalytic centre of water splitting. Here we report the crystal structure of photosystem II at a resolution of 1.9 Å. From our electron density map, we located all of the metal atoms of the Mn4CaO5 cluster, together with all of their ligands. We found that five oxygen atoms served as oxo bridges linking the five metal atoms, and that four water molecules were bound to the Mn4CaO5 cluster; some of them may therefore serve as substrates for dioxygen formation. We identified more than 1,300 water molecules in each photosystem II monomer. Some of them formed extensive hydrogen-bonding networks that may serve as channels for protons, water or oxygen molecules. The determination of the high-resolution structure of photosystem II will allow us to analyse and understand its functions in great detail.

S1-State Model of the O2-Evolving Complex of Photosystem II

We introduce a quantum mechanics/molecular mechanics model of the oxygen-evolving complex of photosystem II in the S(1) Mn(4)(IV,III,IV,III) state, where Ca(2+) is bridged to manganese centers by the carboxylate moieties of D170 and A344 on the basis of the new X-ray diffraction (XRD) model recently reported at 1.9 Å resolution. The model is also consistent with high-resolution spectroscopic data, including polarized extended X-ray absorption fine structure data of oriented single crystals. Our results provide refined intermetallic distances within the Mn cluster and suggest that the XRD model most likely corresponds to a mixture of oxidation states, including species more reduced than those observed in the catalytic cycle of water splitting.

Location of chloride and its possible functions in oxygen-evolving photosystem II revealed by X-ray crystallography

The chloride ion, Cl−, is an essential cofactor for oxygen evolution of photosystem II (PSII) and is closely associated with the Mn4Ca cluster. Its detailed location and function have not been identified, however. We substituted Cl− with a bromide ion (Br−) or an iodide ion (I−) in PSII and analyzed the crystal structures of PSII with Br− and I− substitutions. Substitution of Cl− with Br− did not inhibit oxygen evolution, whereas substitution of Cl− with I− completely inhibited oxygen evolution, indicating the efficient replacement of Cl− by I−. PSII with Br− and I− substitutions were crystallized, and their structures were analyzed. The results showed that there are 2 anion-binding sites in each PSII monomer; they are located on 2 sides of the Mn4Ca cluster at equal distances from the metal cluster. Anion-binding site 1 is close to the main chain of D1-Glu-333, and site 2 is close to the main chain of CP43-Glu-354; these 2 residues are coordinated directly with the Mn4Ca cluster. In addition, site 1 is located in the entrance of a proton exit channel. These results indicate that these 2 Cl− anions are required to maintain the coordination structure of the Mn4Ca cluster as well as the proposed proton channel, thereby keeping the oxygen-evolving complex fully active.

Structure of the catalytic, inorganic core of oxygen-evolving photosystem II at 1.9 Å resolution.

The catalytic center for photosynthetic water-splitting consists of 4 Mn atoms and 1 Ca atom and is located near the lumenal surface of photosystem II. So far the structure of the Mn(4)Ca-cluster has been studied by a variety of techniques including X-ray spectroscopy and diffraction, and various structural models have been proposed. However, its exact structure is still unknown due to the limited resolution of crystal structures of PSII achieved so far, as well as possible radiation damages that might have occurred. Very recently, we have succeeded in solving the structure of photosystem II at 1.9 Å, which yielded a detailed picture of the Mn(4)CaO(5)-cluster for the first time. In the high resolution structure, the Mn(4)CaO(5)-cluster is arranged in a distorted chair form, with a cubane-like structure formed by 3 Mn and 1 Ca, 4 oxygen atoms as the distorted base of the chair, and 1 Mn and 1 oxygen atom outside of the cubane as the back of the chair. In addition, four water molecules were associated with the cluster, among which, two are associated with the terminal Mn atom and two are associated with the Ca atom. Some of these water molecules may therefore serve as the substrates for water-splitting. The high resolution structure of the catalytic center provided a solid basis for elucidation of the mechanism of photosynthetic water splitting. We review here the structural features of the Mn(4)CaO(5)-cluster analyzed at 1.9 Å resolution, and compare them with the structures reported previously.

Theoretical illumination of water-inserted structures of the CaMn4O5 cluster in the S2 and S3 states of oxygen-evolving complex of photosystem II: full geometry optimizations by B3LYP hybrid density functional.

Full geometry optimizations of several inorganic model clusters, CaMn(4)O(4)XYZ(H(2)O)(2) (X, Y, Z = H(2)O, OH(-) or O(2-)), by the use of the B3LYP hybrid density functional theory (DFT) have been performed to illuminate plausible molecular structures of the catalytic site for water oxidation in the S(0), S(1), S(2) and S(3) states of the Kok cycle for the oxygen-evolving complex (OEC) of photosystem II (PSII). Optimized geometries obtained by the energy gradient method have revealed the degree of symmetry breaking of the unstable three-center Mn(a)-X-Mn(d) bond in CaMn(4)O(4)XYZ(H(2)O)(2). The right-elongated (R) Mn(a)-X···Mn(d) and left-elongated (L) Mn(a)···X-Mn(d) structures appear to occupy local minima on a double-well potential for several key intermediates in these states. The effects of insertion of one extra water molecule to the vacant coordination site, Mn(d) (Mn(a)), for R (L) structures have also been examined in detail. The greater stability of the L-type structure over the R-type has been concluded for key intermediates in the S(2) and S(3) states. Implications of the present DFT structures are discussed in relation to previous DFT and related results, together with recent X-ray diffraction results for model compounds of cubane-like OEC cluster of PSII.

Structure of Sr-substituted photosystem II at 2.1 Å resolution and its implications in the mechanism of water oxidation

Oxygen-evolving complex of photosystem II (PSII) is a tetra-manganese calcium penta-oxygenic cluster (Mn4CaO5) catalyzing light-induced water oxidation through several intermediate states (S-states) by a mechanism that is not fully understood. To elucidate the roles of Ca2+ in this cluster and the possible location of water substrates in this process, we crystallized Sr2+-substituted PSII from Thermosynechococcus vulcanus, analyzed its crystal structure at a resolution of 2.1 Å, and compared it with the 1.9 Å structure of native PSII. Our analysis showed that the position of Sr was moved toward the outside of the cubane structure of the Mn4CaO5-cluster relative to that of Ca2+, resulting in a general elongation of the bond distances between Sr and its surrounding atoms compared with the corresponding distances in the Ca-containing cluster. In particular, we identified an apparent elongation in the bond distance between Sr and one of the two terminal water ligands of Ca2+, W3, whereas that of the Sr-W4 distance was not much changed. This result may contribute to the decrease of oxygen evolution upon Sr2+-substitution, and suggests a weak binding and rather mobile nature of this particular water molecule (W3), which in turn implies the possible involvement of this water molecule as a substrate in the O-O bond formation. In addition, the PsbY subunit, which was absent in the 1.9 Å structure of native PSII, was found in the Sr-PSII structure.

Structural-functional role of chloride in photosystem II.

Chloride binding in photosystem II (PSII) is essential for photosynthetic water oxidation. However, the functional roles of chloride and possible binding sites, during oxygen evolution, remain controversial. This paper examines the functions of chloride based on its binding site revealed in the X-ray crystal structure of PSII at 1.9 Å resolution. We find that chloride depletion induces formation of a salt bridge between D2-K317 and D1-D61 that could suppress the transfer of protons to the lumen.

Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography

PsbY is one of the low molecular mass subunits of oxygen‐evolving photosystem II (PSII). Its location, however, has not been identified in the current crystal structure of PSII. We constructed a PsbY‐deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the crystal structure of the mutant PSII dimer. The results obtained showed that PsbY is located in the periphery of PSII close to the α‐ and β‐subunits of cytochrome b559, which corresponded to an unassigned helix in the 3.7 Å structure of T. vulcanus or helix X2 in the 3.0 Å structure of T. elongatus. Our results also indicated that the C‐terminal loop of PsbY is protruded toward the stromal side, instead of the lumenal side predicted previously.

Photosystem II does not possess a simple excitation energy funnel: time-resolved fluorescence spectroscopy meets theory.

The experimentally obtained time-resolved fluorescence spectra of photosystem II (PS II) core complexes, purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus, at 5–180 K are compared with simulations. Dynamic localization effects of excitons are treated implicitly by introducing exciton domains of strongly coupled pigments. Exciton relaxations within a domain and exciton transfers between domains are treated on the basis of Redfield theory and generalized Förster theory, respectively. The excitonic couplings between the pigments are calculated by a quantum chemical/electrostatic method (Poisson-TrEsp). Starting with previously published values, a refined set of site energies of the pigments is obtained through optimization cycles of the fits of stationary optical spectra of PS II. Satisfactorily agreement between the experimental and simulated spectra is obtained for the absorption spectrum including its temperature dependence and the linear dichroism spectrum of PS II core complexes (PS II-CC). Furthermore, the refined site energies well reproduce the temperature dependence of the time-resolved fluorescence spectrum of PS II-CC, which is characterized by the emergence of a 695 nm fluorescence peak upon cooling down to 77 K and the decrease of its relative intensity upon further cooling below 77 K. The blue shift of the fluorescence band upon cooling below 77 K is explained by the existence of two red-shifted chlorophyll pools emitting at around 685 and 695 nm. The former pool is assigned to Chl45 or Chl43 in CP43 (Chl numbering according to the nomenclature of Loll et al. Nature2005, 438, 1040) while the latter is assigned to Chl29 in CP47. The 695 nm emitting chlorophyll is suggested to attract excitations from the peripheral light-harvesting complexes and might also be involved in photoprotection.

Distribution of the Cationic State over the Chlorophyll Pair of the Photosystem II Reaction Center

The reaction center chlorophylls a (Chla) of photosystem II (PSII) are composed of six Chla molecules including the special pair Chla P(D1)/P(D2) harbored by the D1/D2 heterodimer. They serve as the ultimate electron abstractors for water oxidation in the oxygen-evolving Mn(4)CaO(5) cluster. Using the PSII crystal structure analyzed at 1.9 Å resolution, the redox potentials of P(D1)/P(D2) for one-electron oxidation (E(m)) were calculated by considering all PSII subunits and the protonation pattern of all titratable residues. The E(m)(Chla) values were calculated to be 1015-1132 mV for P(D1) and 1141-1201 mV for P(D2), depending on the protonation state of the Mn(4)CaO(5) cluster. The results showed that E(m)(P(D1)) was lower than E(m)(P(D2)), favoring localization of the charge of the cationic state more on P(D1). The P(D1)(•+)/P(D2)(•+) charge ratio determined by the large-scale QM/MM calculations with the explicit PSII protein environment yielded a P(D1)(•+)/P(D2)(•+) ratio of ~80/~20, which was found to be due to the asymmetry in electrostatic characters of several conserved D1/D2 residue pairs that cause the E(m)(P(D1))/E(m)(P(D2)) difference, e.g., D1-Asn181/D2-Arg180, D1-Asn298/D2-Arg294, D1-Asp61/D2-His61, D1-Glu189/D2-Phe188, and D1-Asp170/D2-Phe169. The larger P(D1)(•+) population than P(D2)(•+) appears to be an inevitable fate of the intact PSII that possesses water oxidation activity.