Detection of an intermediary, protonated water cluster in photosynthetic oxygen evolution

Abstract

In photosynthesis, photosystem II evolves oxygen from water by the accumulation of photooxidizing equivalents at the oxygenevolving complex (OEC). The OEC is a Mn 4 CaO 5 cluster, and its sequentially oxidized states are termed the S n states. The darkstable state is S 1 , and oxygen is released during the transition from S 3 to S 0 . In this study, a laser flash induces the S 1 to S 2 transition, which corresponds to the oxidation of Mn(III) to Mn(IV). A broad infrared band, at 2,880 cm, is produced during this transition. Experiments using ammonia and 2 H 2 O assign this band to a cationic cluster of internal water molecules, termed "W 5 + ." Observation of the W 5 + band is dependent on the presence of calcium, and flash dependence is observed. These data provide evidence that manganese oxidation during the S 1 to S 2 transition results in a coupled proton transfer to a substrate-containing, internal water cluster in the OEC hydrogen-bonded network. Internal proton transfer reactions play important catalytic roles in many integral membrane proteins. In these enzymes, including bacteriorhodopsin, light-driven or redox-coupled proton-transfer reactions lead to the production of a transmembrane, electrochemical gradient. Amino acid side chains often participate in the acid/base chemistry that occurs in proton-transfer pathways. However, internal bound water clusters can also play essential roles as proton donors or acceptors (reviewed in ref. 1).In photosystem II (PSII), proton transfer contributes to the generation of a transmembrane potential, and chemical protons are released from the substrate, water, during the light-driven reactions that produce molecular oxygen (2). PSII is a complex membrane protein consisting of both integral, membrane-spanning subunits and extrinsic subunits (3). A monomeric unit of PSII consists of at least 20 distinct protein subunits, which are composed of 17 integral subunits and 3 extrinsic polypeptides (4, 5). The primary subunits that make up the reaction center and bind most of the redox-active cofactors are D1, D2, CP43, and CP47. The light-induced electron transfer pathway in the reaction center involves the dimeric chlorophyll (chl) donor, P 680 , and accessory chl molecules. One light-induced charge separation oxidizes the primary donor, P 680 , and reduces a bound plastoquinone acceptor, Q A . P 680 + oxidizes a tyrosine residue, YZ, Y161 of the D1 polypeptide, which is a powerful oxidant. YZ• oxidizes the oxygen-evolving complex (OEC) on each photoinduced charge separation (reviewed in ref. 6).The OEC is a Mn 4 CaO 5 cluster ( Fig. 1 A, Inset) (5). Oxygen release from the OEC fluctuates with period four (7). The OEC cycles through five sequentially oxidized states, called the S n states. A single flash given to a dark-adapted sample (S 1 state) generates the S 2 state (Fig. 1A), which corresponds to the oxidation of Mn(III) to Mn(IV) (8). Subsequent flashes advance the remaining manganese ions to higher oxidation states, with an accompanying deprotonation of two bound wat...

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

“…Second, spectra were then normalized to the intensity of the ferricyanide and ferrocyanide bands (2116 and 2038 cm Ϫ1 , data not shown), which reflect any small changes in the amount of charge separation in the preparations. These methods have been used previously for PSII spectra (70,71,84) and give control double difference spectra ( Fig. 5F) with flat baselines and no vibrational bands, as expected.…”

“…7, A-D). Oxygen evolution occurs on the first reaction cycle at this temperature, but the sample cannot carry out a second enzymatic cycle, under these flash conditions, possibly due to a limitation in water diffusion or a conformational gate (84). There is evidence that the steady-state rate of electron transfer under continuous illumination is slower in the FT-IR samples, when compared with a room temperature oxygen assay ("Experimental Procedures").…”

“…The FT-IR data, presented here, suggest that the amide bonds of PsbO are sensors for the functional, extended hydrogen-bonded water network in the OEC. Spectral differences observed at 263 K and 277 K may be due to inhibited diffusion of water or to a conformational gate at 263 K, under the conditions employed for flash excitation (84).…”

Orientation of non-spherical protonated water clusters revealed by infrared absorption dichroism

“…Our experimental approach is general and can be used to study the orientation of protonated water clusters also in other membrane proteins, particularly those that involve proton translocation, like, e.g., photosystem II 53 , photosynthetic reaction center 54 , 55 , archaerhodopsin-3 56 , and cytochrome c oxidase 57 . Apart from proteins, we expect similar effects also in other systems that contain water clusters, for example, in inverted hexagonal lipid phases, where water forms hexagonally ordered parallel cylinders, or in lipid lamellar phases, where water forms thin slabs 58 .…”

Water is an active matrix of life for cell and molecular biology

“…3C). In one stage of the oxygen-generating cycle, a hydrogen-bonded cluster of water molecules at this site acts as a catalytic proton acceptor, storage site, and donor (63)(64)(65): an example of bound water serving as a reactive chemical substrate.…”