Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

The mechanism and kinetics of electron transfer in isolated D1͞ D2-cyt b559 photosystem (PS) II reaction centers (RCs) and in intact PSII cores have been studied by femtosecond transient absorption and kinetic compartment modeling. For intact PSII, a component of Ϸ1.5 ps reflects the dominant energy-trapping kinetics from the antenna by the RC. A 5.5-ps component reflects the apparent lifetime of primary charge separation, which is faster by a factor of 8 -12 than assumed so far. The 35-ps component represents the apparent lifetime of formation of a secondary radical pair, and the Ϸ200-ps component represents the electron transfer to the Q A acceptor. In isolated RCs, the apparent lifetimes of primary and secondary charge separation are Ϸ3 and 11 ps, respectively. It is shown (i) that pheophytin is reduced in the first step, and (ii) that the rate constants of electron transfer in the RC are identical for PSII cores and for isolated RCs. We interpret the first electron transfer step as electron donation from the primary electron donor Chl acc D1. Thus, this mechanism, suggested earlier for isolated RCs at cryogenic temperatures, is also operative in intact PSII cores and in isolated RCs at ambient temperature. The effective rate constant of primary electron transfer from the equilibrated RC* excited state is 170 -180 ns ؊1 , and the rate constant of secondary electron transfer is 120 -130 ns ؊1 .charge separation ͉ photosynthesis ͉ ultrafast spectroscopy ͉ D1͞D2-cytb559 ͉ femtosecond absorption P hotosystem (PS) II cores, whose structure has recently been determined to a resolution of 3.5-3.2 Å (1-3), consist of the antenna polypeptides CP43 and CP47, which carry 13 and 16 chlorophyll (Chl) a molecules, respectively. They contain furthermore the D1͞D2-cyt b559 reaction center (RC) polypeptides, which bind the pigments of the electron transfer chain [four Chls, two pheophytins (Pheo), and two quinones] and two additional antenna Chls (the so-called Chl z D1 and Chl z D2 molecules). The isolated RC (D1-D2-cyt b559 ) lacks the quinone acceptors and is thus only able to create a short-lived radical pair (RP) (see review in ref. 4).There exists presently no agreement on the mechanism of the primary events of energy and electron transfer in the isolated RC complex (see refs. 4-6 for recent reviews). Early studies suggested an apparent Ϸ3-ps charge separation lifetime in the RC at room temperature (7,8) in agreement with later studies (9, 10). Andrizhiyevskaya et al. (11) recently also proposed a model with an Ϸ3-ps charge separation. A somewhat slower charge separation of Ϸ8 ps has been reported by Wasielewski and coworkers (12), whereas more recent data from the same group were interpreted in terms of a 2-to 5-ps charge separation time (13). Substantially shorter charge separation times of 1 ps (14) and 0.4 ps (at 240 K) have been reported by Groot et al. (15). At the other extreme, a 1 order of magnitude longer charge separation time of Ϸ21 ps has been suggested by Klug and coworkers (16,17). Probably the largest ...

The assignment of the two substrate water sites of the tetramanganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn 4 O 5 Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522-3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55-60]}; (ii) ammonia displaces a water ligand to the outer manganese (Mn A4 -W1); and (iii) as W1 is trans to O5, ammonia binding elongates the Mn A4 -O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.PSII | OEC | water oxidizing complex | water-oxidation | Mn cluster I n oxygenic photosynthesis, light-driven water splitting is catalyzed by the oxygen-evolving complex (OEC) of the membrane bound, pigment-protein complex photosystem II (PSII). The OEC consists of an inorganic tetra-manganese penta-oxygen calcium (Mn 4 O 5 Ca) cluster (1-3) and the nearby redox-active tyrosine residue Y Z (D1-Tyr161) that couples electron transfer from the Mn 4 O 5 Ca cluster to P680, the photo-oxidant of PSII. The cluster resembles a "distorted chair", where the base is formed by an oxygen-bridged (μ-oxo) cuboidal Mn 3 O 4 Ca unit (1) (Fig. 1A). The fourth Mn (Mn A4 ) is located outside of the cuboidal unit and is linked via a μ-oxo-bridged ligation (O4) to one of its corners (Mn B3 ). A second linkage between the outer Mn and the cube is provided by a fifth oxygen O5. The Mn 4 O 5 Ca cluster is also held together by six carboxylate ligands and has only one directly coordinating nitrogen ligand, D1-His332 (Fig. 1B).The OEC cycles through a series of five intermediate states that are known as S states (4) (Fig. 1A): S 0 , S 1 (dark stable), S 2 , S 3 , and S 4 (not yet isolated), where the subscript refers to the number of oxidizing equivalents stored in the OEC through successive electron withdrawals by Y Z • . In the 1.9-Å resolution structure, the S state of the cluster was assigned to be S 1 (1). However, this is unlikely as all Mn-Mn, Mn-Ca, and Mn-O/N distances of the crystal structure are ∼...

Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and 15 N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

Biogenesis of photosystem II (PSII), nature's water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-heme iron with glutamate, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction.