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A 3-year IUPAC project Systems Thinking in Chemistry for Sustainability: Toward 2030 and Beyond (STCS 2030+, IUPAC Project #2020-014-3-050) [1] launched in late 2020 is breaking important new ground in addressing chemistry’s orientations, roles, and responsibilities in the 21st Century and helping to map out implications for chemistry education, research, and practice. In taking on this ambitious task, STCS 2030+ draws on expertise available within IUPAC’s own structures, as a project co-sponsored by three IUPAC standing committees: the Committee on Chemistry Education (CCE), the Committee on Chemistry and Industry (COCI) and the Interdivisional Committee on Green Chemistry for Sustainable Development (ICGCSD). The project is also working with other organizations, such as the International Organization for Chemical Sciences in Development (IOCD), which is a co-supporter, and involves collaborators with individuals from organizations that include the Stockholm Resilience Centre [2], the American Chemical Society (ACS) Green Chemistry Institute [3], the International Year of Basic Sciences for Sustainable Development (IYBSSD 2022-23) [4], and chemistry educators and chemical industry from around the world.

What is the history and justification of the claim that chemistry is “the central science”? Are our students able to see and appreciate such centrality of the knowledge of chemistry in the curriculum as well as in its broader interconnections with other fields of study and with societal issues? In this editorial for the special issue Reimagining Chemistry Education: Systems Thinking, and Green and Sustainable Chemistry, we, as guest editors, highlight the case made in the collective contributions to the special issue that systems thinking, including green and sustainable chemistry, shows promise as an approach to help chemistry students zoom out from detailed and fragmented disciplinary content to obtain a more holistic view of chemistry and its integral connection to earth and societal systems. Indeed, we ask the following question: How can we as a community of chemists and chemistry educators live up to the claim of being practitioners of the central science if we do not equip our students to engage in systems thinking? We further invite the community to make use of and build upon the contributions in this special issue and participate in a paradigm change in chemistry education that will help prepare our students to be citizens and scientists who are better equipped to deal with the grand challenges in the world.

The International Union of Pure & Applied Chemistry (IUPAC) launched a global project in 2017 to infuse systems thinking into chemistry education, motivated in part by the desire to help equip chem...

Graphical representations are an important tool used to model abstract processes in fields such as chemistry. Successful interpretation of a graph involves a combination of mathematical expertise and discipline-specific content to reason about the relationship between the variables and to describe the phenomena represented. In this work, we studied students’ graphical reasoning as they responded to a chemical kinetics prompt. Qualitative data was collected and analyzed for a sample of 70 students through the use of an assessment involving short-answer test items administered in a first-year, non-majors chemistry course at a Swedish university. The student responses were translated from Swedish to English and subsequently coded to analyze the chemical and mathematical ideas students attributed to the graph. Mathematical reasoning and ideas related to covariation were analyzed using graphical forms and the shape thinking perspective of graphical reasoning. Student responses were further analyzed by focusing on the extent to which they integrated chemistry and mathematics. This was accomplished by conceptualizing modeling as discussing mathematical narratives, characterizing how students described the “story” communicated by the graph. Analysis provided insight into students’ understanding of mathematical models of chemical processes.

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H(2)O(2) detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1–5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro. Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H(2)O(2). However, no iron oxidation with O(2) as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

The tyrosine residue D2-Tyr160 (Tyr(D)) in photosystem II (PSII) can be oxidized through charge equilibrium with the oxygen evolving complex in PSII. The kinetics of the electron transfer from Tyr( ...

The Journal of Chemical Education announces a call for papers for an upcoming special issue on Reimagining Chemistry Education: Systems Thinking, and Green and Sustainable Chemistry.

Journal of biological inorganic chemistry 24(6), 863 - 877 (2019). doi:10.1007/s00775-019-01697-8, Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of ribonucleotide reductase (RNR), class Id, is dependent on a Mn2(IV,III) cofactor for catalysis. Class I RNRs consist of a substrate-binding component (NrdA) and a metal-containing radical-generating component (NrdB). Herein we utilize a combination of EPR spectroscopy and enzyme assays to underscore the enzymatic relevance of the Mn2(IV,III) cofactor in class Id NrdB from Facklamia ignava. Once formed, the Mn2(IV,III) cofactor confers enzyme activity that correlates well with cofactor quantity. Moreover, we present the X-ray structure of the apo- and aerobically Mn-loaded forms of the homologous class Id NrdB from Leeuwenhoekiella blandensis, revealing a dimanganese centre typical of the subclass, with a tyrosine residue maintained at distance from the metal centre and a lysine residue projected towards the metals. Structural comparison of the apo- and metal-loaded forms of the protein reveals a refolding of the loop containing the conserved lysine and an unusual shift in the orientation of helices within a monomer, leading to the opening of a channel towards the metal site. Such major conformational changes have not been observed in NrdB proteins before. Finally, in vitro reconstitution experiments reveal that the high-valent manganese cofactor is not formed spontaneously from oxygen, but can be generated from at least two different reduced oxygen species, i.e. H$_2$O$_2$ and superoxide (O$_2^{·−}$ ). Considering the observed differences in the efficiency of these two activating reagents, we propose that the physiologically relevant mechanism involves superoxide., Published by Springer, New York

The use and interpretation of graphs pose significant challenges to the learner, but also open up opportunities for developing skills in combining both chemical and mathematical knowledge in proble ...

This project is motivated by an increasing demand from public bodies and research funding agencies for outreach and public engagement, believing this to be a path towards enhanced public understanding, recruitment and research with impact. Yet many STEM (Science, Technology, Engineering and Mathematics) graduates and professionals lack the appropriate communication skills required to engage with the public. To address some of these issues, the project RACE (RAw Communication and Engagement) was jointly initiated by universities and industrial partners across Europe. Through the design and implementation of adaptable modules incorporating content knowledge, scientific communication and public engagement skills, this international project aims to equip students and researchers alike to ground their work within the wider global society and communicate their research with public audiences. A key feature is the direct incorporation of actual public engagement activities into RACE modules, for the mutual benefit of participants and wider society. Given this, the chapter will take a practitioner perspective to shed light on the inner workings of a modular scientific communication course. Core issues of engagement, bridging theory and practice, evaluation and collaboration are all highlighted through reflections and research data from implementing the RACE programme as a Ph.D. summer school with doctoral-level students. The chapter is concluded by bringing forward core teaching and learning protocols that are integral to running the RACE programme.

Dps proteins (DNA-binding proteins from starved cells) have been found to detoxify H2O2. At their catalytic centers, the ferroxidase center (FOC), Dps proteins utilize Fe2+ to reduce H2O2 and therefore play an essential role in the protection against oxidative stress and maintaining iron homeostasis. Whereas most bacteria accommodate one or two Dps, there are five different Dps proteins in Nostoc punctiforme, a phototrophic and filamentous cyanobacterium. This uncommonly high number of Dps proteins implies a sophisticated machinery for maintaining complex iron homeostasis and for protection against oxidative stress. Functional analyses and structural information on cyanobacterial Dps proteins are rare, but essential for understanding the function of each of the NpDps proteins. In this study, we present the crystal structure of NpDps4 in its metal-free, iron- and zinc-bound forms. The FOC coordinates either two iron atoms or one zinc atom. Spectroscopic analyses revealed that NpDps4 could oxidize Fe2+ utilizing O2, but no evidence for its use of the oxidant H2O2 could be found. We identified Zn2+ to be an effective inhibitor of the O2-mediated Fe2+ oxidation in NpDps4. NpDps4 exhibits a FOC that is very different from canonical Dps, but structurally similar to the atypical one from DpsA of Thermosynechococcus elongatus. Sequence comparisons among Dps protein homologs to NpDps4 within the cyanobacterial phylum led us to classify a novel FOC class: the His-type FOC. The features of this special FOC have not been identified in Dps proteins from other bacterial phyla and it might be unique to cyanobacterial Dps proteins.

The filamentous cyanobacterium Nostoc punctiforme ATCC 29133 produces hydrogen via nitrogenase in heterocysts upon onset of nitrogen-fixing conditions. N. punctiforme expresses concomitantly the uptake hydrogenase HupSL, which oxidizes hydrogen in an effort to recover some of the reducing power used up by nitrogenase. Eliminating uptake activity has been employed as a strategy for net hydrogen production in N. punctiforme (Lindberg et al., Int. J. Hydrogen Energy, 2002, 27, 1291-1296). However, nitrogenase activity wanes within a few days. In the present work, we modify the proximal iron-sulfur cluster in the hydrogenase small subunit HupS by introducing the designed mutation C12P in the fusion protein f-HupS for expression in E. coli (Raleiras et al., J. Biol. Chem., 2013, 288, 18345-18352), and in the full HupSL enzyme for expression in N. punctiforme. C12P f-HupS was investigated by EPR spectroscopy and found to form a new paramagnetic species at the proximal cluster site consistent with a [4Fe-4S] to [3Fe-4S] cluster conversion. The new cluster has the features of an unprecedented mixed-coordination [3Fe-4S] metal center. The mutation was found to produce stable protein in vitro, in silico and in vivo. When C12P HupSL was expressed in N. punctiforme, the strain had a consistently higher hydrogen production than the background [capital Delta]hupSL mutant. We conclude that the increase in hydrogen production is due to the modification of the proximal iron-sulfur cluster in HupS, leading to a turn of the electron flow in the enzyme.

This Forum article addresses some of the issues raised in the article by Ying-Syuan Huang and Anila Asghar's paper entitled: Science education reform in Confucian learning cultures: teachers' perspectives on policy and practice in Taiwan. An attempt is made to highlight the need for a more nuanced approach in considering the Confucian education tradition and its compatibility with education reforms. In particular, the article discusses issues concerning the historical development of the Confucian education tradition, challenges in reform implementation that are in reality tradition-independent, as well as opportunities and points of convergence that the Confucian education tradition presents that can in fact be favorable to implementation of reform-based pedagogies.

Protein engineering is a powerful tool to modify e.g. protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium Synechocystis PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in Synechocystis PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both in vivo and in vitro resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in Synechocystis PCC 6803.

Chemistry education focused on learning outcomes is increasingly practiced, providing new opportunities for international comparisons. The interest in intended learning outcomes and constructive alignment has grown in many parts of the world due to both research in higher education and political decisions. In an International Union of Pure and Applied Chemistry (IUPAC) project, we have developed a method of using critical evaluation of learning outcomes and descriptors at international, national, and institutional levels to enhance learner-centered chemistry education. This method is process-focused, aimed at learning by sharing and comparing practices around the world. Three overarching documents for the chemistry bachelor from the USA, Europe and Australia were compared. The differences were found to be more in style than in content. A tool for self-analysis was constructed to evaluate how learning outcomes for courses and modules are linked to each other and to learning outcomes for educational programs and how the expected learning outcomes can be aligned with learning activities and assessment. We conclude that the method can be used to elucidate the correspondence between learning outcomes at different levels, and the constructive alignment between learning outcomes, learning activities and assessment. The process gives new perspectives and shared knowledge. Chemistry education may need to be different depending on local considerations, and awareness of these differences is of value for further development.

Water is clearly important for the functioning of Photosystem II (PSII). Apart from being the very substrate that needs to be transported in this water oxidation enzyme, water is also vital for the transport of protons to and from the catalytic center as well as other important co-factors and key residues in the enzyme. The latest crystal structural data of PSII have enabled detailed analyses of the location and possible function of water molecules in the enzyme. Significant progress has also been made recently in the investigation of channels and pathways through the protein complex. Through these studies, the mechanistic significance of water for PSII is becoming increasingly clear. An overview and discussion of key aspects of the current research on water in PSII is presented here. The role of water in three other systems (aquaporin, bacteriorhodopsin and cytochrome P450) is also outlined to illustrate further points concerning the central significance that water can have, and potential applications of these ideas for continued research on PSII. It is advocated that water be seen as an integral part of the protein and far from a mere solvent.

There are many reasons chemistry education varies among nations and institutions: cultural traditions, societal expectations, socio-economic factors, and educational resources, to name just a few. At the university level there is also a dependence on the earlier educational system, as well as the research profile of the particular institution. In an increasingly globalized world, however, graduate mobility across international boundaries is more relevant than ever. In order to promote high quality chemistry education, comparisons and communication at both national and international levels are important for designing or improving programs. Such comparisons can raise the awareness of what to change and what to hold on to.

Cryogenic illumination of Photosystem II (PSII) can lead to the trapping of the metastable radical Y(Z)(•), the radical form of the redox-active tyrosine residue D1-Tyr161 (known as Y(Z)). Magnetic interaction between this radical and the CaMn(4) cluster of PSII gives rise to so-called split electron paramagnetic resonance (EPR) signals with characteristics that are dependent on the S state. We report here the observation and characterization of a split EPR signal that can be directly induced from PSII centers in the S(2) state through visible light illumination at 10 K. We further show that the induction of this split signal takes place via a Mn-centered mechanism, in the same way as when using near-infrared light illumination [Koulougliotis, D., et al. (2003) Biochemistry 42, 3045-3053]. On the basis of interpretations of these results, and in combination with literature data for other split signals induced under a variety of conditions (temperature and light quality), we propose a unified model for the mechanisms of split signal induction across the four S states (S(0), S(1), S(2), and S(3)). At the heart of this model is the stability or instability of the Y(Z)(•)(D1-His190)(+) pair that would be formed during cryogenic oxidation of Y(Z). Furthermore, the model is closely related to the sequence of transfers of protons and electrons from the CaMn(4) cluster during the S cycle and further demonstrates the utility of the split signals in probing the immediate environment of the oxygen-evolving center in PSII.

The advent of oxygenic photosynthesis through water oxidation by photosystem II (PSII) transformed the planet, ultimately allowing the evolution of aerobic respiration and an explosion of ecological diversity. The importance of this enzyme to life on Earth has ironically been paralleled by the elusiveness of a detailed understanding of its precise catalytic mechanism. Computational investigations have in recent years provided more and more insights into the structural and mechanistic details that underlie the workings of PSII. This review will present an overview of some of these studies, focusing on those that have aimed at elucidating the mechanism of water oxidation at the CaMn4 cluster in PSII, and those exploring the features of the structure and dynamics of this enzyme that enable it to catalyse this energetically demanding reaction. This article is part of a Special Issue entitled: Photosystem II.

Metalloradical EPR signals have been found in intact Photosystem II at cryogenic temperatures. They reflect the light-driven formation of the tyrosine Z radical (Y(Z)) in magnetic interaction with the CaMn(4) cluster in a particular S state. These so-called split EPR signals, induced at cryogenic temperatures, provide means to study the otherwise transient Y(Z) and to probe the S states with EPR spectroscopy. In the S(0) and S(1) states, the respective split signals are induced by illumination of the sample in the visible light range only. In the S(3) state the split EPR signal is induced irrespective of illumination wavelength within the entire 415-900nm range (visible and near-IR region) [Su, J. H., Havelius, K. G. V., Ho, F. M., Han, G., Mamedov, F., and Styring, S. (2007) Biochemistry 46, 10703-10712]. An important question is whether a single mechanism can explain the induction of the Split S(3) signal across the entire wavelength range or whether wavelength-dependent mechanisms are required. In this paper we confirm that the Y(Z) radical formation in the S(1) state, reflected in the Split S(1) signal, is driven by P680-centered charge separation. The situation in the S(3) state is different. In Photosystem II centers with pre-reduced quinone A (Q(A)), where the P680-centered charge separation is blocked, the Split S(3) EPR signal could still be induced in the majority of the Photosystem II centers using both visible and NIR (830nm) light. This shows that P680-centered charge separation is not involved. The amount of oxidized electron donors and reduced electron acceptors (Q(A)(-)) was well correlated after visible light illumination at cryogenic temperatures in the S(1) state. This was not the case in the S(3) state, where the Split S(3) EPR signal was formed in the majority of the centers in a pathway other than P680-centered charge separation. Instead, we propose that one mechanism exists over the entire wavelength interval to drive the formation of the Split S(3) signal. The origin for this, probably involving excitation of one of the Mn ions in the CaMn(4) cluster in Photosystem II, is discussed.

The redox-active tyrosine residue (Y-Z) plays a crucial role in the mechanism of the water oxidation. Metalloradical electron paramagnetic resonance (EPR) signals reflecting the light-induced Y-Z c ...

Even prior to the publication of the crystal structures for photosystem II (PSII), it had already been suggested that water, O(2) and H(+) channels exist in PSII to achieve directed transport of these molecules, and to avoid undesirable side reactions. Computational efforts to uncover these channels and investigate their properties are still at early stages, and have so far only been based on the static PSII structure. The rationale behind the proposals for such channels and the computer modelling studies thus far are reviewed here. The need to take the dynamic protein into account is then highlighted with reference to the specific issues and techniques applicable to the simulation of each of the three channels. In particular, lessons are drawn from simulation studies on other protein systems containing similar channels.

The long-lived, light-induced radical Y-D(.) of the Tyr161 residue in the D2 protein of Photosystem 11 (PSII) is known to magnetically interact with the CaMn4 cluster, situated similar to 30 angstrom away. In this study we report a transient step-change increase in YD EPR intensity upon the application of a single laser flash to S, state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of YD by the OEC in the S-2 state, as a consequence of the single laser flash turnover. The post-flash decay reflected S-2 -> S-1 back-turnover, as confirmed by their correlations with independent measurements of S-2 multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using YD as a probe. (c) 2006 Elsevier B.Z. All rights reserved. (Less)

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.

Illuminating a photosystem II sample at low temperatures (here 5-10 K) yields so-called split signals detectable with continuous wave-electron paramagnetic resonance (CW-EPR). These signals reflect the oxidized, deprotonated radical of D1-Tyr161 (YZ(•)) in a magnetic interaction with the CaMn4 cluster in a particular S state. The intensity of the split EPR signals are affected by the addition of the water substrate analogue methanol. This was previously shown by the induction of split EPR signals from the S1, S3, and S0 states [Su, J.-H. et al. (2006) Biochemistry 45, 7617-7627.]. Here, we use two split EPR signals induced from photosystem II trapped in the S2 state to further probe the binding of methanol in an S state dependent manner. The signals are induced with either visible or near-infrared light illumination provided at 5-10 K where methanol cannot bind or unbind from its site. The results imply that the binding of methanol not only changes the magnetic properties of the CaMn4 cluster but also the hydrogen bond network in the oxygen evolving complex (OEC), thereby affecting the relative charge of the S2 state. The induction mechanisms for the two split EPR signals are different resulting in two different redox states, S2YZ(•) and S1YZ(•) respectively. The two states show different methanol dependence for their induction. This indicates the existence of two binding sites for methanol in the CaMn4 cluster. It is proposed that methanol binds to MnA with high affinity and to MnD with lower affinity. The molecular nature and S-state dependence of the methanol binding to each respective site are discussed.

A putative H+ exit pathway consisting of residues surrounding a dynamically stable water chain leading from the CaMn4 cluster towards the lumen was studied using molecular dynamics (MD) simulations. A number of residues that were previously shown to be important for H+ transport in vivo were found to interact with this water chain, consistent with the Grotthuss mechanism. Furthermore, MD data from PSII mutated in silico elucidated possible structural and electrostatic roles of other residues not previously regarded as directly involved in H+ transport. These results could be correlated with literature data of in vivo mutants, and an earlier proposal of certain residues acting as a localised buffer for receiving released H+. Overall, comparisons of our MD results of in silico mutants with in vivo mutagenesis data have revealed some unifying themes in the roles played by different residues involved in H+ transport.

During oxygenic photosynthesis, the oxidation of water to molecular oxygen and a proton is catalysed by the enzyme Photosystem II (PSII). The reaction takes place at a CaMn4 cluster that is found within the PSII protein matrix, shielded from the external environment. It has been proposed that dedicated channels and pathways exist in the protein complex for the passage of the substrate and product molecules to and from this active site. In this chapter, the rationales for the possible existence of such pathways are considered, and the various biochemical, spectroscopic and computational studies for identifying their possible locations and functions are reviewed. The different approaches and techniques employed and their influences on the respective results and conclusions are also compared and discussed.

Five nitrogen fixing cyanobacterial strains have been found to contain PsaB2, an additional and divergent gene copy for the Photosystem I reaction center protein PsaB. In all five species the divergent gene, psaB2, is located separately from the normal psaAB operon in the genome. The protein, PsaB2, was recently identified in heterocysts of Nostoc punctiforme sp. strain PCC 73102. 12 conserved amino acid replacements and one insertion, were identified by a multiple sequence alignment of several PsaB2 and PsaB1 sequences. Several, including an inserted glutamine, are located close to the iron-sulfur cluster FX in the electron transfer chain. By homology modeling, using the Photosystem I crystal structure as template, we have found that the amino acid composition in PsaB2 will introduce changes in critical parts of the Photosystem I protein structure. The changes are close to FX and the phylloquinone (PhQ) in the B-branch, indicating that the electron transfer properties most likely will be affected. We suggest that the divergent PsaB2 protein produces an alternative Photosystem I reaction center with different structural and electron transfer properties. Some interesting physiologcial consequences that this can have for the function of Photosystem I in heterocysts, are discussed.

Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of Q(A)((-)) is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (

A split EPR signal can be induced at 5 K in Photosystem II in the S3-state by light in the range of 400–900 nm. To investigate if the same mechanism is involved in the signal induction in the full spectral range we compared the properties of the S3 signal induced by 830 nm light or white light. Our results indicate that the same mechanism is responsible for the formation of the “Split S3” signal in the whole spectral range. The mechanism of the “Split S3” signal is not P680 driven but is instead driven by manganese excitation.

We have analyzed S-transition dependent miss fractions in the S-cycle using the S1 and S3 split EPR signals together with the S2 state multiline EPR signal. We demonstrate that quantification of the S1 and S3 states through the use of the split EPR signals. This provides a novel probe to the S-cycle efficiency. Our analysis shows that, in the S2 → S3 transition, misses involve 35% of the photosystem II centres at 0–2°C. By contrast, our results strongly indicate that there are no misses in the S1 → S2 transition at this low temperature. Consequently misses during the S-cycle are strongly S-state dependent.

EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn(4) cluster are known in all S states except S(4). Many signals are insufficiently understood and the S(0), S(1), and S(3) states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S(0), S(1), and S(3) states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S(0), split S(1), split S(3) and S(2) state multiline EPR signals. We demonstrate that quantification of the S(1), S(3) and S(0) states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S(1) --S(2) transition proceeds without misses following a saturating flash at 1 degrees C, whilst substantial misses occur in the S(2) --S(3) transition following the second flash.

The interaction EPR split signals from photosystem II (PSII) have been reported from the S0, S1, and S3 states. The signals are induced by illumination at cryogenic temperatures and are proposed to reflect the magnetic interaction between YZ• and the Mn 4Ca cluster. We have investigated the formation spectra of these split EPR signals induced in PSII enriched membranes at 5 K using monochromatic laser light from 400 to 900 nm. We found that the formation spectra of the split S0, split S1, and split S3 EPR signals were quite similar, but not identical, between 400 and 690 nm, with maximum formation at 550 nm. The major deviations were found between 440 and 480 nm and between 580 and 680 nm. In the regions around 460 and 680 nm the amplitudes of the formation spectra were 25-50% of that at 550 nm. A similar formation spectrum was found for the S2-state multiline EPR signal induced at 0°C. In general, the formation spectra of these signals in the visible region resemble the reciprocal of the absorption spectra of our PSII membranes. This reflects the high chlorophyll concentration necessary for the EPR measurements which mask the spectral properties of other absorbing species. No split signal formation was found by the application of infrared laser illumination between 730 and 900 nm from PSII in the S0 and S1 states. However, when such illumination was applied to PSII membranes poised in the S 3 state, formation of the split S3 EPR signal was observed with maximum formation at 740 nm. The quantum yield was much less than in the visible region, but the application of intensive illumination at 830 nm resulted in accumulation of the signal to an amplitude comparable to that obtained with illumination with visible light. The split S3 EPR signal induced by NIR light was much more stable at 5 K (no observable decay within 60 min) than the split S3 signal induced by visible light (50% of the signal decayed within 30 min). The split S3 signals induced by each of these light regimes showed the same EPR spectral features and microwave power saturation properties, indicating that illumination of PSII in the S3 state by visible light or by NIR light produces a similar configuration of YZ• and the Mn4Ca cluster. (Less)

Given the tightly packed environment of Photosystem II (PSII), channels are expected to exist within the protein to allow the movement of small molecules to and from the oxygen evolving centre. In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S1-, S3-, and S0-signals [Su et al. (2006) Biochemistry 45, 7617–7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn4 cluster, only 3Mn and 4Mn were accessible to methanol. Combining this observation with spectroscopic data in the literature, we propose that 3Mn is the ion to which methanol binds. Furthermore, by calculating solvent contact surfaces for water, we found analogous and more extensive water accessible channels within PSII. On the basis of their structure, orientation, and electrostatic properties, we propose functional assignments of these channels as passages for substrate water access to the CaMn4 cluster, and for the exit of O2 and H+ that are released during water oxidation. Finally, we discuss the possible existence of a gating mechanism for the control of substrate water access to the CaMn4 cluster, based on the observation of a gap within the channel system that is formed by Ca2+ and several mechanistically very significant residues in the vicinity of the cluster.

Methanol binds to the CaMn4 cluster in photosystem II (PSII). Here we report the methanol dependence of the split EPR signals originating from the magnetic interaction between the CaMn4 cluster and the Y(Z)* radical in PSII which are induced by illumination at 5 K. We found that the magnitudes of the "split S1" and "split S3" signals induced in the S1 and S3 states of PSII centers, respectively, are diminished with an increase in the methanol concentration. The methanol concentrations at which half of the respective spectral changes had occurred ([MeOH](1/2)) were 0.12 and 0.57%, respectively. By contrast, the "split S0" signal induced in the S0 state is broadened, and its amplitude is enhanced. [MeOH](1/2) for this change was found to be 0.54%. We discuss these observations with respect to the location and nature of the methanol binding site. Furthermore, by comparing this behavior with methanol effects reported for other EPR signals in the different S states, we propose that the observed methanol-dependent changes in the split S1 and split S0 EPR signals are caused by an increase in the extent of magnetic coupling within the cluster.

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.