The First State in the Catalytic Cycle of the Water-Oxidizing Enzyme: Identification of a Water-Derived µ-Hydroxo Bridge.

Nature's water-splitting catalyst, an oxygen-bridged tetramanganese calcium (Mn4O5Ca) complex, sequentially activates two substrate water molecules generating molecular O2. Its reaction cycle is composed of five intermediate (Si) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S0. Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of 18O-labeled water incorporation into the product O2 is slow, on a millisecond to second time scale depending on the S state. Here, we demonstrate that the Mn4O5Ca complex poised in the S0 state contains an exchangeable hydroxo bridge. On the basis of a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15561]. This oxygen species is the most probable candidate for the slowly exchanging substrate water in the S0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.

The Cyanobacterial Photoactive Orange Carotenoid Protein Is an Excellent Singlet Oxygen Quencher.

Cyanobacteria have developed a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers under high-light conditions. The photoactive orange carotenoid protein (OCP) is essential in this mechanism as a light sensor and energy quencher. When OCP is photoactivated by strong blue-green light, it is able to dissipate excess energy as heat by interacting with phycobilisomes. As a consequence, charge separation and recombination leading to the formation of singlet oxygen diminishes. Here, we demonstrate that OCP has another essential role. We observed that OCP also protects Synechocystis cells from strong orange-red light, a condition in which OCP is not photoactivated. We first showed that this photoprotection is related to a decrease of singlet oxygen concentration due to OCP action. Then, we demonstrated that, in vitro, OCP is a very good singlet oxygen quencher. By contrast, another carotenoid protein having a high similarity with the N-terminal domain of OCP is not more efficient as a singlet oxygen quencher than a protein without carotenoid. Although OCP is a soluble protein, it is able to quench the singlet oxygen generated in the thylakoid membranes. Thus, OCP has dual and complementary photoprotective functions as an energy quencher and a singlet oxygen quencher.

Acetate in mixotrophic growth medium affects photosystem II in Chlamydomonas reinhardtii and protects against photoinhibition.

Chlamydomonas reinhardtii is a photoautotrophic green alga, which can be grown mixotrophically in acetate-supplemented media (Tris-acetate-phosphate). We show that acetate has a direct effect on photosystem II (PSII). As a consequence, Tris-acetate-phosphate-grown mixotrophic C. reinhardtii cultures are less susceptible to photoinhibition than photoautotrophic cultures when subjected to high light. Spin-trapping electron paramagnetic resonance spectroscopy showed that thylakoids from mixotrophic C. reinhardtii produced less (1)O2 than those from photoautotrophic cultures. The same was observed in vivo by measuring DanePy oxalate fluorescence quenching. Photoinhibition can be induced by the production of (1)O2 originating from charge recombination events in photosystem II, which are governed by the midpoint potentials (Em) of the quinone electron acceptors. Thermoluminescence indicated that the Em of the primary quinone acceptor (QA/QA(-)) of mixotrophic cells was stabilised while the Em of the secondary quinone acceptor (QB/QB(-)) was destabilised, therefore favouring direct non-radiative charge recombination events that do not lead to (1)O2 production. Acetate treatment of photosystem II-enriched membrane fragments from spinach led to the same thermoluminescence shifts as observed in C. reinhardtii, showing that acetate exhibits a direct effect on photosystem II independent from the metabolic state of a cell. A change in the environment of the non-heme iron of acetate-treated photosystem II particles was detected by low temperature electron paramagnetic resonance spectroscopy. We hypothesise that acetate replaces the bicarbonate associated to the non-heme iron and changes the environment of QA and QB affecting photosystem II charge recombination events and photoinhibition.

Charge separation in photosystem II: a comparative and evolutionary overview.

Our current understanding of the PSII reaction centre owes a great deal to comparisons to the simpler and better understood, purple bacterial reaction centre. Here we provide an overview of the similarities with a focus on charge separation and the electron acceptors. We go on to discuss some of the main differences between the two kinds of reaction centres that have been highlighted by the improving knowledge of PSII. We attempt to relate these differences to functional requirements of water splitting. Some are directly associated with that function, e.g. high oxidation potentials, while others are associated with regulation and protection against photodamage. The protective and regulatory functions are associated with the harsh chemistry performed during its normal function but also with requirements of the enzyme while it is undergoing assembly and repair. Key aspects of PSII reaction centre evolution are also addressed. This article is part of a Special Issue entitled: Photosystem II.

A high redox potential form of cytochrome c550 in photosystem II from Thermosynechococcus elongatus.

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ∼300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.

Effects of formate binding on the quinone-iron electron acceptor complex of photosystem II.

EPR was used to study the influence of formate on the electron acceptor side of photosystem II (PSII) from Thermosynechococcus elongatus. Two new EPR signals were found and characterized. The first is assigned to the semiquinone form of Q(B) interacting magnetically with a high spin, non-heme-iron (Fe²(+), S=2) when the native bicarbonate/carbonate ligand is replaced by formate. This assignment is based on several experimental observations, the most important of which were: (i) its presence in the dark in a significant fraction of centers, and (ii) the period-of-two variations in the concentration expected for Q(B)(•-) when PSII underwent a series of single-electron turnovers. This signal is similar but not identical to the well-know formate-modified EPR signal observed for the Q(A)(•-)Fe²(+) complex (W.F.J. Vermaas and A.W. Rutherford, FEBS Lett. 175 (1984) 243-248). The formate-modified signals from Q(A)(•-)Fe²(+) and Q(B)(•-)Fe²(+) are also similar to native semiquinone-iron signals (Q(A)(•-)Fe²(+)/Q(B)(•-)Fe²(+)) seen in purple bacterial reaction centers where a glutamate provides the carboxylate ligand to the iron. The second new signal was formed when Q(A)(•-) was generated in formate-inhibited PSII when the secondary acceptor was reduced by two electrons. While the signal is reminiscent of the formate-modified semiquinone-iron signals, it is broader and its main turning point has a major sub-peak at higher field. This new signal is attributed to the Q(A)(•-)Fe²(+) with formate bound but which is perturbed when Q(B) is fully reduced, most likely as Q(B)H2 (or possibly Q(B)H(•-) or Q(B)(²â€¢-)). Flash experiments on formate-inhibited PSII monitoring these new EPR signals indicate that the outcome of charge separation on the first two flashes is not greatly modified by formate. However on the third flash and subsequent flashes, the modified Q(A)(•-)Fe²(+)Q(B)H2 signal is trapped in the EPR experiment and there is a marked decrease in the quantum yield of formation of stable charge pairs. The main effect of formate then appears to be on Q(B)H2 exchange and this agrees with earlier studies using different methods.

Semiquinone-iron complex of photosystem II: EPR signals assigned to the low-field edge of the ground state doublet of QA•-Fe2+ and QB•-Fe2+.

The quinone-iron complex of the electron acceptor complex of Photosystem II was studied by EPR spectroscopy in Thermosynechococcus elongatus. New g ∼ 2 features belonging to the EPR signal of the semiquinone forms of the primary and secondary quinone, i.e., Q(A)(•-)Fe(2+) and Q(B)(•-)Fe(2+), respectively, are reported. In previous studies, these signals were missed because they were obscured by the EPR signal arising from the stable tyrosyl radical, TyrD(•). When the TyrD(•) signal was removed, either by chemical reduction or by the use of a mutant lacking TyrD, the new signals dominated the spectrum. For Q(A)(•-)Fe(2+), the signal was formed by illumination at 77 K or by sodium dithionite reduction in the dark. For Q(B)(•-)Fe(2+), the signal showed the characteristic period-of-two variations in its intensity when generated by a series of laser flashes. The new features showed relaxation characteristics comparable to those of the well-known features of the semiquinone-iron complexes and showed a temperature dependence consistent with an assignment to the low-field edge of the ground state doublet of the spin system. Spectral simulations are consistent with this assignment and with the current model of the spin system. The signal was also present in Q(B)(•-)Fe(2+) in plant Photosystem II, but in plants, the signal was not detected in the Q(A)(•-)Fe(2+) state.

High and low potential forms of the QA quinone electron acceptor in Photosystem II of Thermosynechococcus elongatus and spinach.

The redox potential of Q(A) in Photosystem II (PSII) from Thermosynechococcus elongatus was titrated monitoring chlorophyll fluorescence. A high potential form (E(m)=+60 ± 25 mV) was found in the absence of Mn(4)Ca, the active site for water oxidation. The low potential form (E(m)=-60 ± 48 mV), which is difficult to measure in conventional titration experiments, could be "locked in" by cross-linking the active enzyme. This indicates that the presence of Mn(4)Ca is relayed to the quinone site by significant structural changes in the protein. The presence of high and low potential forms agrees with what has been seen in plants, algae from our lab and in T. elongatus (Shibamoto et al., Biochemistry 48 (2009) 10682-10684). In the latter work, the potentials of Q(A) were shifted to lower potentials compared to other measurements. The redox potential of Q(A) in Mn-depleted PSII from spinach was titrated in the presence of redox mediators and the midpoint potential was shifted by 80 mV towards a more negative value compared to titrations without mediators. The lower values of the midpoint potential of the (Q(A)/Q(A)(-)) redox couple in the literature could be due to a perturbation due to a specific mediator.