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

Todellista asiakasarvoa luomassa – myynnistä arvomyyntiin : case Mediamaisteri Oy

Tämän opinnäytetyön tavoite oli case-yritys Mediamaisteri Oy:n B2B-myyntiprosessin kehittäminen. Tarkoitus oli hyödyntää palvelumuotoilua lisäämään asiakasymmärrystä ja tukemaan asiakkaiden kokeman arvon muodostumista sekä positiivisten asiakaskokemusten syntymistä myyntiprosessin alkuvaiheen asiakaskohtaamisissa. Lisäksi tarkoitus oli kiteyttää empiirisen aineiston pohjalta saatu tieto asiakasprofiileiksi, joita voidaan hyödyntää asiakaslähtöisten ratkaisujen suunnittelussa heti asiakkuuden alusta ja ensimmäisestä myyntitapaamisesta lähtien. Opinnäytetyön tutkimuksellisen osuuden toteuttamisen pohjana on kvalitatiivinen tutkimusote ja palvelumuotoilun prosessi. Tutkimuskohde tarkennettiin kuvaamalla Mediamaisteri Oy:n asiakkuuden alkuvaiheen palvelupolku, josta rajattiin tutkimuksen kohteeksi ensimmäinen asiakastapaaminen ja sitä edeltävä aika, jolloin asiakas kiinnostuu yrityksen palveluista ja ensikontakti yritykseen tapahtuu. Tutkimusaineisto muodostuu kuudesta Mediamaisteri Oy:n yritysasiakkaan haastattelusta, jotka toteutettiin virtuaalisesti teemahaastattelun muodossa. Aineiston analysoinnissa hyödynnettiin induktiivista sisällönanalyysiä. Opinnäytetyön teoreettinen viitekehys rakentuu B2B-myynnin, asiakasarvon muodostumisen ja positiivisen asiakaskokemuksen teorioista. Opinnäytetyön empiirisessä osassa kuvataan yksityiskohtaisesti työn toteuttaminen ja toteutuksen vaiheittainen eteneminen. Opinnäytetyön tuloksien mukaan Mediamaisteri Oy:n yritysasiakkaan positiivinen asiakaskokemus palvelujen hankintavaiheessa ja ensimmäisen asiakastapaamisen aikana koostuu myyjäyrityksen koetusta osaamisesta ja ammattitaidosta, palveluasenteesta, asiakaslähtöisyydestä, vuorovaikutustaidoista, yhteistyötaidoista, avoimuuden ja luottamuksen tunteen herättämisestä ja projektinhallinnasta. Opinnäytetyön tutkimusaineiston pohjalta kerätty asiakastieto kiteytettiin Mediamaisteri Oy:n yritysasiakkaan asiakasprofiileiksi, joita voidaan tulevaisuudessa hyödyntää yrityksen asiakaslähtöisen myyntiprosessin kehittämisessä suuntaamaan toimintaa kohti todellista asiakasarvoa asiakkuuden alkuvaiheesta alkaen. Voidaan todeta, että opinnäytetyön tuloksilla on yhteyttä opinnäytetyön viitekehyksessä esitettyyn aikaisempaan teoreettiseen tietoon B2B-myynnistä, asiakasarvon muodostumisesta ja positiivisesta asiakaskokemuksesta. Opinnäytetyön tuloksissa ilmeni, että asiakasymmärryksen tavoittelu jää palvelujen hankintavaiheessa ja ensimmäisessä asiakastapaamisessa usein pinnalliselle tasolle. Jatkotutkimus etenkin vuorovaikutustaitojen merkityksestä B2B-myyntiprosessin alussa ja ensimmäisessä B2B-myyntitapaamisessa arvonluonnin ja arvomyynnin edellyttämän asiakasymmärryksen saavuttamiseksi on suositeltavaa.The aim of this thesis was to develop the B2B sales process of the case company Mediamaisteri Oy. The purpose was to utilize service design to promote the customer insight and to support the formation of the customer value and positive customer experiences in customer touchpoints during the early stages of the sales process. In addition, the purpose was to crystallize the information obtained from the empirical data into customer profiles that can be used to design customer-oriented solutions right from the beginning of a customer relationship and the initial B2B sales meeting. The basis for carrying out the empirical part of the thesis was the qualitative research and the process of service design. The research subject was defined by describing the business client’s customer journey in the beginning of the customer relationship with Mediamaisteri Oy, and by focusing to the initial B2B sales meeting and the time when the client became interested in the company's services and made the first contact. The research material consisted of six interviews of Mediamaisteri Oy's business clients, made as online theme interviews. The material was analyzed by utilizing inductive content analysis. The theoretical framework of the thesis was based on the theories of B2B sales, customer value creation and positive customer experience. In the empirical part of the thesis, the implementation of the work and the steps of the process are described in detail. The findings of the thesis indicate that the positive customer experience of a Mediamaisteri business client during the procurement phase and the initial B2B sales meeting consists of proven expertise and professionalism, service attitude, customer orientation, interaction skills, co-operation skills, openness and trust, and the project management. The research data collected was crystallized into the business client profiles of Mediamaisteri Oy, which can be used in the future to develop the customer-oriented sales process of the company in order to direct the business towards the real customer value starting right from the beginning of the customer relationship. It can be concluded that the results of the thesis are related to the previous theoretical knowledge of B2B sales, customer value creation and a positive customer experience presented in the theoretical framework of the thesis. The results of the thesis showed that the pursuit of customer understanding during the procurement phase and the initial B2B sales meeting often remains superficial. Further research on the significance of interaction skills in the beginning of the sales process and at the initial B2B sales meeting is desirable to achieve value creation and value-based customer insight

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

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 QB(center dot-) 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)(center dot-)Fe(2+) complex (W.F.J. Vermaas and A.W. Rutherford, FEBS Lett. 175 (1984) 243248). The formate-modified signals from Q(A)(center dot-)Fe(2+) and Q(B)(center dot-)Fe(2+) are also similar to native semiquinone-iron signals Q(A)(center dot-)Fe(2+)/Q(B)(center dot-)Fe(2+)) seen in purple bacterial reaction centers where a glutamate provides the carboxylate ligand to the iron. The second new signal was formed when QA(center dot-) 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)(center dot-)Fe(2+) with formate bound but which is perturbed when Q(B) is fully reduced, most likely as Q(B)H(2) (or possibly Q(B)H(center dot-) or Q(B)H(2 center dot-)). 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)(center dot-)Fe(2+)Q(B)H(2) 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)H(2) exchange and this agrees with earlier studies using different method

Apport de la fibre optique pour l’étude des ancrages passifs scellés au rocher en milieu fracturé

International audienceLa fracturation des massifs rocheux impacte le comportement des ancrages passifs scellés au rocher utilisés pour la gestion du risque rocheux. Nous présentons les résultats d’essais d’arrachement instrumentés par fibre optique réalisés sur trois sites présentant des niveaux de fracturation variables. Ces résultats couplés avec de l’endoscopie permettent de mieux comprendre les sollicitations en profondeur

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