Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages towards the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria

Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting.

In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.This work was supported by the U.K. Engineering and Physical Sciences Research Council (EP/H00338X/2 to E.R. and EP/G037221/1, nanoDTC, to D.M.), the UK Biology and Biotechnological Sciences Research Council (BB/K002627/1 to A.W.R. and BB/K010220/1 to E.R.), a Marie Curie Intra-European Fellowship (PIEF-GA-2013-625034 to C.Y.L), a Marie Curie International Incoming Fellowship (PIIF-GA-2012-328085 RPSII to J.J.Z) and the CEA and the CNRS (to J.C.F.C.). A.W.R. holds a Wolfson Merit Award from the Royal Society.This is the final version of the article. It first appeared from ACS Publications via http://dx.doi.org/10.1021/jacs.5b0373

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

Mechanism of proton-coupled quinone reduction in Photosystem II.

Photosystem II uses light to drive water oxidation and plastoquinone (PQ) reduction. PQ reduction involves two PQ cofactors, Q(A) and Q(B), working in series. Q(A) is a one-electron carrier, whereas Q(B) undergoes sequential reduction and protonation to form Q(B)H(2). Q(B)H(2) exchanges with PQ from the pool in the membrane. Based on the atomic coordinates of the Photosystem II crystal structure, we analyzed the proton transfer (PT) energetics adopting a quantum mechanical/molecular mechanical approach. The potential-energy profile suggests that the initial PT to Q(B)(•-) occurs from the protonated, D1-His252 to Q(B)(•)(-) via D1-Ser264. The second PT is likely to occur from D1-His215 to Q(B)H(-) via an H-bond with an energy profile with a single well, resulting in the formation of Q(B)H(2) and the D1-His215 anion. The pathway for reprotonation of D1-His215(-) may involve bicarbonate, D1-Tyr246 and water in the Q(B) site. Formate ligation to Fe(2+) did not significantly affect the protonation of reduced Q(B), suggesting that formate inhibits Q(B)H(2) release rather than its formation. The presence of carbonate rather than bicarbonate seems unlikely because the calculations showed that this greatly perturbed the potential of the nonheme iron, stabilizing the Fe(3+) state in the presence of Q(B)(•-), a situation not encountered experimentally. H-bonding from D1-Tyr246 and D2-Tyr244 to the bicarbonate ligand of the nonheme iron contributes to the stability of the semiquinones. A detailed mechanistic model for Q(B) reduction is presented

Charge separation in Photosystem II: A comparative and evolutionary overview

AbstractOur 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

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