Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel-production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese-calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reactioncycle model of photosynthetic water oxidation. In the S 2 → S 3 transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (Y Z ox ). The rate-determining elementary step (τ, approximately 30 μs at 20°C) in the long-distance proton relocation toward the protein-water interface is characterized by a high activation energy (E a ¼ 0.46 AE 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S 0 → S 1 transition are similar (τ, approximately 100 μs; E a ¼ 0.34 AE 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to Y Z ox . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

The intricate orchestration of electron transfer (ET) and proton transfer (PT) at the Mn4CaOn-cluster of photosystem II (PSII) is mechanistically pivotal but clearly insufficiently understood. Preparations of PSII membrane particles were investigated using a kinetically competent and sensitive method, photothermal beam deflection (PBD), to monitor apparent volume changes of the PSII protein. Driven by nanosecond laser flashes, the PSII was synchronously stepped through its water-oxidation cycle involving four (semi)stable states (S0, S1, S2, and S3) and minimally three additional transiently formed intermediates. The PBD approach was optimized as compared to our previous experiments, resulting in superior signal quality and resolution of more reaction steps. Now seven transitions were detected and attributed, according to the H/D-exchange, temperature, and pH effects on their time constants, to ET or PT events. The ET steps oxidizing the Mn4CaOn cluster in the S2 → S3 and S0 → S1 transitions, a biphasic PT prior to the O2-evolving reaction, as well as the reoxidation of the primary quinone acceptor (QA(-)) at the PSII acceptor side were detected for the first time by PBD. The associated volume changes involve (i) initial formation of charged groups resulting in contraction assignable to electrostriction, (ii) volume contraction explainable by reduced metal-ligand distances upon manganese oxidation, and (iii) charge-compensating proton removal resulting in volume expansion due to electrostriction reversal. These results support a reaction cycle of water oxidation exhibiting alternate ET and PT steps. An extended kinetic scheme for the O2-evolving S3 ⇒ S0 transition is proposed, which includes crucial structural and protonic events.

The Mn complex of photosystem II (PSII) cycles through 4 semi-stable states (S(0) to S(3)). Laser-flash excitation of PSII in the S(2) or S(3) state induces processes with time constants around 350ns, which have been assigned previously to energetic relaxation of the oxidized tyrosine (Y(Z)(ox)). Herein we report monitoring of these processes in the time domain of hundreds of nanoseconds by photoacoustic (or 'optoacoustic') experiments involving pressure-wave detection after excitation of PSII membrane particles by ns-laser flashes. We find that specifically for excitation of PSII in the S(2) state, nuclear rearrangements are induced which amount to a contraction of PSII by at least 30Å(3) (time constant of 350ns at 25°C; activation energy of 285+/-50meV). In the S(3) state, the 350-ns-contraction is about 5 times smaller whereas in S(0) and S(1), no volume changes are detectable in this time domain. It is proposed that the classical S(2)=>S(3) transition of the Mn complex is a multi-step process. The first step after Y(Z)(ox) formation involves a fast nuclear rearrangement of the Mn complex and its protein-water environment (~350ns), which may serve a dual role: (1) The Mn- complex entity is prepared for the subsequent proton removal and electron transfer by formation of an intermediate state of specific (but still unknown) atomic structure. (2) Formation of the structural intermediate is associated (necessarily) with energetic relaxation and thus stabilization of Y(Z)(ox) so that energy losses by charge recombination with the Q(A)(-) anion radical are minimized. The intermediate formed within about 350ns after Y(Z)(ox) formation in the S(2)-state is discussed in the context of two recent models of the S(2)=>S(3) transition of the water oxidation cycle. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: From Natural to Artificial.

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890–1903). Recent PBD results on formation of the Q⁻(A)/Y(•+)(Z) radical pair suggest a value of ΔH similar to the free energy change, ΔG, of -540±40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S₃−−>S₀, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q⁻(A)/Y(•+)(Z) formation (<1 μs) and the S₃−−>S₀ transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 ų per PSII unit) in the S₃−−>S₀ transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O₂ and proton release. The observed volume changes show that the S₃−−>S₀ transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.

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