Photodamage to the oxygen evolving complex of photosystem II by visible light.

Light damages photosynthetic machinery, primarily photosystem II (PSII), and it results in photoinhibition. A new photodamage model, the two-step photodamage model, suggests that photodamage to PSII initially occurs at the oxygen evolving complex (OEC) by light energy absorbed by manganese and that the PSII reaction center is subsequently damaged by light energy absorbed by photosynthetic pigments due to the limitation of electrons to the PSII reaction center. However, it is still uncertain whether this model is applicable to photodamage to PSII under visible light as manganese absorbs visible light only weakly. In the present study, we identified the initial site of photodamage to PSII upon illumination of visible light using PSII membrane fragments isolated from spinach leaves. When PSII samples were exposed to visible light in the presence of an exogenous electron acceptor, both PSII total activity and the PSII reaction centre activity declined due to photodamage. The supplemental addition of an electron donor to the PSII reaction centre alleviated the decline of the reaction centre activity but not the PSII total activity upon the light exposure. Our results demonstrate that visible light damages OEC prior to photodamage to the PSII reaction center, consistent with two-step photodamage model.

Photo-oxidation of tyrosine in a bio-engineered bacterioferritin 'reaction centre'-a protein model for artificial photosynthesis.

The photosynthetic reaction centre (RC) is central to the conversion of solar energy into chemical energy and is a model for bio-mimetic engineering approaches to this end. We describe bio-engineering of a Photosystem II (PSII) RC inspired peptide model, building on our earlier studies. A non-photosynthetic haem containing bacterioferritin (BFR) from Escherichia coli that expresses as a homodimer was used as a protein scaffold, incorporating redox-active cofactors mimicking those of PSII. Desirable properties include: a di-nuclear metal binding site which provides ligands for bivalent metals, a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin and presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. Light-induced electron transfer from proximal tyrosine residues to the photo-oxidised ZnCe6(•+), in the modified BFR reconstituted with both ZnCe6 and Mn(II), is presented. Three site-specific tyrosine variants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in the engineered system. The results indicate that: presence of bound Mn(II) is necessary to observe tyrosine oxidation in all BFR variants; Y45 the most important tyrosine as an immediate electron donor to the oxidised ZnCe6(•+) and that Y25 and Y58 are both redox-active in this system, but appear to function interchangebaly. High-resolution (2.1Å) crystal structures of the tyrosine variants show that there are no mutation-induced effects on the overall 3-D structure of the protein. Small effects are observed in the Y45F variant. Here, the BFR-RC represents a protein model for artificial photosynthesis.

The role of nano-sized manganese oxides in the oxygen-evolution reactions by manganese complexes: towards a complete picture.

Eighteen Mn complexes with N-donor and carboxylate ligands have been synthesized and characterized. Three Mn complexes among them are new and are reported for the first time. The reactions of oxygen evolution in the presence of oxone (2KHSO5·KHSO4·K2SO4) and cerium(iv) ammonium nitrate catalyzed by these complexes are studied and characterized by UV-visible spectroscopy, X-ray diffraction spectrometry, dynamic light scattering, Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, transmission electron microscopy, scanning electron microscopy, membrane-inlet mass spectrometry and electrochemistry. Some of these complexes evolve oxygen in the presence of oxone as a primary oxidant. CO2 and MnO4(-) are other products of these reactions. Based on spectroscopic studies, the true catalysts for oxygen evolution in these reactions are different. We proposed that for the oxygen evolution reactions in the presence of oxone, the true catalysts are both high valent Mn complexes and Mn oxides, but for the reactions in the presence of cerium(iv) ammonium nitrate, the active catalyst is most probably a Mn oxide.

Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues.

The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular organisms, liberating free energy needed to catalyze a plethora of enzymatic reactions. Measuring the isotope signatures of (16)O and (18)O during O2 reduction can provide insights into both kinetic and equilibrium isotope effects. However, current methods to measure O2 isotope signatures are time-consuming and disruptive. This paper describes the application of membrane inlet mass spectrometry to determine the oxygen isotope discrimination of a range of O2-consuming reactions, providing a rapid and convenient method for determining these values. A survey of oxygenase and oxidase reactions provides new insights into previously uncharacterized amino acid oxidase enzymes. Liquid and gas phase measurements show the ease of assays using this approach for purified enzymes, biological extracts and intact tissues.

Network of hydrogen bonds near the oxygen-evolving Mn(4)CaO(5) cluster of photosystem II probed with FTIR difference spectroscopy.

We previously provided experimental evidence that an extensive network of hydrogen bonds exists near the oxygen-evolving Mn4CaO5 cluster in photosystem II and that elements of this network form part of a dominant proton-egress pathway leading from the Mn4CaO5 cluster to the thylakoid lumen. The evidence was based on (i) the elimination of the same ν(C═O) mode of a protonated carboxylate group in the S2-minus-S1 FTIR difference spectrum of wild-type PSII core complexes from the cyanobacterium Synechocystis sp. PCC 6803 by the mutations D1-E65A, D2-E312A, and D1-E329Q and (ii) the substantial decrease in the efficiency of the S3 to S0 transition caused by the mutations D1-D61A, D1-E65A, and D2-E312A. The eliminated ν(C═O) mode corresponds to an unidentified carboxylate group whose pKa value decreases in response to the increased charge that develops on the Mn4CaO5 cluster during the S1 to S2 transition. In the current study, we have extended our work to include the ν(C═O) regions of other Sn+1-minus-Sn FTIR difference spectra and to additional mutations of residues inferred to participate in networks of hydrogen bonds near the Mn4CaO5 cluster or leading from the Mn4CaO5 cluster to the thylakoid lumen. Our data suggest that a different carboxylate group has its pKa value increased during the S2 to S3 transition and that a third carboxylate group experiences a change in its environment during the S0 to S1 transition. The pKa values that shift during the S1 to S2 and S2 to S3 transitions appear to be restored during the S3 to S0 transition. The D1-R334A mutation decreases or eliminates the same ν(C═O) modes from the S2-minus-S1 and S3-minus-S2 spectra as mutations D1-E65A, D2-E312A, and D1-E329Q and substantially decreases the efficiency of the S3 to S0 transition. We conclude that D1-R334 participates in the same dominant proton-egress pathway that was identified in our previous study. The D1-Q165E mutation leaves the ν(C═O) region of the S2-minus-S1 FTIR difference spectrum intact, but it eliminates a mode from this region of the S3-minus-S2 spectrum. We conclude that D1-Q165 participates in an extensive network of hydrogen bonds that that extends across the Mn4CaO5 cluster to the D1-E65/D2-E312 dyad and that includes D1-E329 and several water molecules including the W2 and W3 water ligands of the Mn4CaO5 cluster's dangling MnA4 and Ca ions, respectively. The D2-E307Q, D2-D308N, D2-E310Q, and D2-E323Q mutations alter the ν(C═O) regions of none of the FTIR difference spectra. We conclude that these four residues are located far from the three unidentified carboxylate groups that give rise to the ν(C═O) features observed in the FTIR difference spectra.

Participation of glutamate-333 of the D1 polypeptide in the ligation of the Mn4CaO5 cluster in photosystem II.

In the 1.9 Å structural model of photosystem II (PDB: 3ARC), the amino acid residue Glu333 of the D1 polypeptide coordinates to the oxygen-evolving Mn4CaO5 cluster. This residue appears to be highly significant in that it bridges the two Mn ions (Mn(B3) and the "dangling" Mn(A4)) that are also bridged by the oxygen atom O5. This oxygen atom has been proposed to be derived from one of two substrate water molecules and to become incorporated into the product dioxygen molecule during the final step in the catalytic cycle. In addition, the backbone nitrogen of D1-Glu333 interacts directly with a nearby Cl⁻ atom. To further explore the influence of this structurally unique residue on the properties of the Mn4CaO5 cluster, the D1-E333Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, and FTIR difference spectroscopy. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild-type. In addition, the oxygen flash yields exhibited normal period-four oscillations having normal S state parameters, although the yields were lower, indicative of the mutant's lower steady-state dioxygen evolution rate of approximately 30% compared to that of the wild-type. The S1 state Mn-XANES and Mn-EXAFS and S2 state multiline EPR signals of purified D1-E333Q PSII core complexes closely resembled those of wild-type, aside from having lower amplitudes. The S(n+1)-minus-S(n) FTIR difference spectra showed only minor alterations to the carbonyl, amide, and carboxylate stretching regions. However, the mutation eliminated a negative peak at 3663 cm⁻¹ in the weakly H-bonding O-H stretching region of the S2-minus-S1 FTIR difference spectrum and caused an approximately 9 cm⁻¹ downshift of the negative feature in this region of the S1-minus-S0 FTIR difference spectrum. We conclude that fully functional Mn4CaO5 clusters assemble in the presence of the D1-E333Q mutation but that the mutation decreases the yield of assembled clusters and alters the H-bonding properties of one or more water molecules or hydroxide groups that are located on or near the Mn4CaO5 cluster and that either deprotonate or form stronger hydrogen bonds during the S0 to S1 and S1 to S2 transitions.

Temperature modulation of fatty acid profiles for biofuel production in nitrogen deprived Chlamydomonas reinhardtii.

This study investigated the changes in the fatty acid content and composition in the nitrogen-starved Chlamydomonas reinhardtii starchless mutant, BAF-J5, grown at different temperatures. The optimal temperature for vegetative growth under nitrogen sufficient conditions was found to be 32 °C. Shifting temperature from 25 to 32 °C, in conjunction with nitrogen starvation, resulted in BAF-J5 storing the maximum quantity of fatty acid (76% of dry cell weight). Shifting to temperatures lower than 25 °C, reduced the total amount of stored fatty acid content and increased the level of desaturation in the fatty acids. The optimal fatty acid composition for biodiesel was at 32 °C. This study demonstrates how a critical environmental factor, such as temperature, can modulate the amount and composition of fatty acids under nitrogen deprivation and reduce the requirement for costly refining of biofuels.

Transition metal-alkane σ-complexes with oxygen donor co-ligands.

A new family of long-lived alkane σ-complexes of the type (L(OEt))Re(CO)(2)(alkane) [alkane = cyclopentane, cyclohexane, pentane; L(OEt) = cyclopentadienyltris(diethylphosphito)cobaltate(III)] has been observed using both IR and NMR spectroscopies and computationally interrogated with DFT methods. The oxygen-rich coordination spheres makes these complexes perhaps more relevant as models for intermediates in metal oxide mediated hydrocarbon transformations than other known alkane σ-complexes.

Fatty acid profiling of Chlamydomonas reinhardtii under nitrogen deprivation.

The Chlamydomonas reinhardtii starch-less mutant, BAF-J5, was found to store lipids up to 65% of dry cell weight when grown photoheterotrophically and subjected to nitrogen starvation. Fourier transform infrared spectroscopy was used as a high-throughput method for semi-quantitative measurements of protein, carbohydrate and lipid content. The fatty acids of wild-type and starch mutants were identified and quantified by gas chromatography mass spectrometry. C. reinhardtii starch mutants, BAF-J5 and I7, produce significantly elevated levels of 16:0, 18:1(Δ9), 18:2(Δ9,12) and 18:3(Δ9,12,15) fatty acids. Long-chain saturated, mono- and polyunsaturated fatty acids were found under nitrogen starvation. Oleosin-like and caleosin-like genes were identified in the C. reinhardtii genome. However, proteomic analysis of isolated lipid bodies only identified a key lipid droplet associated protein. This study shows it is possible to manipulate algal biosynthetic pathways to produce high levels of lipid that may be suitable for conversion to liquid fuels.

Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II.

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn(4)Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn(4)Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H(2)(18)O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S(3) state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S(1) state Mn-EXAFS spectrum, a slightly altered S(2) state multiline EPR signal, a substantially altered S(2)-minus-S(1) FTIR difference spectrum, and an unusually long lifetime for the S(2) state (>10 h) in a substantial fraction of reaction centers. In contrast, the S(2) state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S(2)-minus-S(1) FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with (15)N and specific labeling with l-[1-(13)C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO(-) group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm(-1) in both the S(1) and S(2) states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S(2) state and that most of these can achieve the S(3) state, but no evidence for advancement beyond the S(3) state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn(4)Ca cluster in the S(2) state, the FTIR and H(2)(18)O exchange data show that the mutation strongly influences other properties of the Mn(4)Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S(1) to S(2) transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S(3) state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn(4)Ca cluster in the S(1) state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S(1) to S(2) transition. The H(2)(18)O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S(3) state.

The evolution of Photosystem II: insights into the past and future.

This article attempts to address the molecular origin of Photosystem II (PSII), the central component in oxygenic photosynthesis. It discusses the possible evolution of the relevant cofactors needed for splitting water into molecular O2 with respect to the following functional domains in PSII: the reaction center (RC), the oxygen evolving complex (OEC), and the manganese stabilizing protein (MSP). Possible ancestral sources of the relevant cofactors are considered, as are scenarios of how these components may have been brought together to produce the intermediate steps in the evolution of PSII. Most importantly, the driving forces that maintained these intermediates for continued adaptation are considered. We then apply our understanding of the evolution of PSII to the bioengineering of a water oxidizing catalyst for utilization of solar energy.

Evidence from FTIR difference spectroscopy of an extensive network of hydrogen bonds near the oxygen-evolving Mn(4)Ca cluster of photosystem II involving D1-Glu65, D2-Glu312, and D1-Glu329.

Analyses of the refined X-ray crystallographic structures of photosystem II (PSII) at 2.9-3.5 A have revealed the presence of possible channels for the removal of protons from the catalytic Mn(4)Ca cluster during the water-splitting reaction. As an initial attempt to verify these channels experimentally, the presence of a network of hydrogen bonds near the Mn(4)Ca cluster was probed with FTIR difference spectroscopy in a spectral region sensitive to the protonation states of carboxylate residues and, in particular, with a negative band at 1747 cm(-1) that is often observed in the S(2)-minus-S(1) FTIR difference spectrum of PSII from the cyanobacterium Synechocystis sp. PCC 6803. On the basis of its 4 cm(-1) downshift in D(2)O, this band was assigned to the carbonyl stretching vibration (C horizontal lineO) of a protonated carboxylate group whose pK(a) decreases during the S(1) to S(2) transition. The positive charge that forms on the Mn(4)Ca cluster during the S(1) to S(2) transition presumably causes structural perturbations that are transmitted to this carboxylate group via electrostatic interactions and/or an extended network of hydrogen bonds. In an attempt to identify the carboxylate group that gives rise to this band, the FTIR difference spectra of PSII core complexes from the mutants D1-Asp61Ala, D1-Glu65Ala, D1-Glu329Gln, and D2-Glu312Ala were examined. In the X-ray crystallographic models, these are the closest carboxylate residues to the Mn(4)Ca cluster that do not ligate Mn or Ca and all are highly conserved. The 1747 cm(-1) band is present in the S(2)-minus-S(1) FTIR difference spectrum of D1-Asp61Ala but absent from the corresponding spectra of D1-Glu65Ala, D2-Glu312Ala, and D1-Glu329Gln. The band is also sharply diminished in magnitude in the wild type when samples are maintained at a relative humidity of

Production and diffusion of chloroplastic H2O2 and its implication to signalling.

Hydrogen peroxide (H(2)O(2)) is recognized as an important signalling molecule. There are two important aspects to this function: H(2)O(2) production and its diffusion to its sites of action. The production of H(2)O(2) by photosynthetic electron transport and its ability to diffuse through the chloroplast envelope membranes has been investigated using spin trapping electron paramagnetic resonance spectroscopy and H(2)O(2)-sensitive fluorescence dyes. It was found that, even at low light intensity, a portion of H(2)O(2) produced inside the chloroplasts can leave the chloroplasts thus escaping the effective antioxidant systems located inside the chloroplast. The production of H(2)O(2) by chloroplasts and the appearance of H(2)O(2) outside chloroplasts increased with increasing light intensity and time of illumination. The amount of H(2)O(2) that can be detected outside the chloroplasts has been shown to be up to 5% of the total H(2)O(2) produced inside the chloroplasts at high light intensities. The fact that H(2)O(2) produced by chloroplasts can be detected outside these organelles is an important finding in terms of understanding how chloroplastic H(2)O(2) can serve as a signal molecule.

The solar action spectrum of photosystem II damage.

The production of oxygen and the supply of energy for life on earth rely on the process of photosynthesis using sunlight. Paradoxically, sunlight damages the photosynthetic machinery, primarily photosystem II (PSII), leading to photoinhibition and loss of plant performance. However, there is uncertainty about which wavelengths are most damaging to PSII under sunlight. In this work we examined this in a simple experiment where Arabidopsis (Arabidopsis thaliana) leaves were exposed to different wavelengths of sunlight by dispersing the solar radiation across the surface of the leaf via a prism. To isolate only the process of photodamage, the repair of photodamaged PSII was inhibited by infiltration of chloramphenicol into the exposed leaves. The extent of photodamage was then measured as the decrease in the maximum quantum yield of PSII using an imaging pulse amplitude modulation fluorometer. Under the experimental light conditions, photodamage to PSII occurred most strongly in regions exposed to ultraviolet (UV) or yellow light. The extent of UV photodamage under incident sunlight would be greater than we observed when one corrects for the optical efficiency of our system. Our results suggest that photodamage to PSII under sunlight is primarily associated with UV rather than photosynthetically active light wavelengths.

On-line mass spectrometry: membrane inlet sampling.

Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O(2) evolution versus O(2) uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H(2) generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and and the experiments that have been conducted with MIMS.

Evidence that D1-His332 in photosystem II from Thermosynechococcus elongatus interacts with the S3-state and not with the S2-state.

Oxygen evolution by Photosystem II (PSII) is catalyzed by a Mn(4)Ca cluster. Thus far, from the crystallographic three-dimensional (3D) structures, seven amino acid residues have been identified as possible ligands of the Mn(4)Ca cluster. Among them, there is only one histidine, His332, which belongs to the D1 polypeptide. The relationships of the D1-His332 amino acid with kinetics and thermodynamic properties of the Mn(4)Ca cluster in the S(2)- and S(3)-states of the catalytic cycle were investigated in purified PSII from Thermosynechococcus elongatus. This was done by examining site-directed D1-His332Gln and D1-His332Ser mutants by a variety of spectroscopic techniques such as time-resolved UV-visible absorption change spectroscopy, cw- and pulse-EPR, thermoluminescence, and measurement of substrate water exchange. Both mutants grew photo-autotrophically and active PSII could be purified. On the basis of the parameters assessed in this work, the D1-His332(Gln, Ser) mutations had no effect in the S(2)-state. Electron spin-echo envelope modulation (ESEEM) spectroscopy also showed that possible interactions between the nuclear spin of the nitrogen(s) of D1-His332 with the electronic spin S = 1/2 of the Mn(4)Ca cluster in the S(2)-state were not detectable and that the D1-His332Ser mutation did not affect the detected hyperfine couplings. In contrast, the following changes were observed in the S(3)-state of the D1-His332 mutants: (1) The redox potential of the S(3)/S(2) couple was slightly increased by < or = 20 meV, (2) The S(3)-EPR spectrum was slightly modified, (3) The D1-His332Gln mutation resulted in a approximately 3 fold decrease of the slow (tightly bound) exchange rate and a approximately 2 fold increase of the fast exchange rate of the water substrate molecules. All these results suggest that the D1-His332 would be more involved in S(3) than in S(2). This could be one element of the conformational changes put forward in the S(2) to S(3) transition.

Photo-catalytic oxidation of a di-nuclear manganese centre in an engineered bacterioferritin 'reaction centre'.

Photosynthesis involves the conversion of light into chemical energy through a series of electron transfer reactions within membrane-bound pigment/protein complexes. The Photosystem II (PSII) complex in plants, algae and cyanobacteria catalyse the oxidation of water to molecular O2. The complexity of PSII has thus far limited attempts to chemically replicate its function. Here we introduce a reverse engineering approach to build a simple, light-driven photo-catalyst based on the organization and function of the donor side of the PSII reaction centre. We have used bacterioferritin (BFR) (cytochrome b1) from Escherichia coli as the protein scaffold since it has several, inherently useful design features for engineering light-driven electron transport. Among these are: (i.) a di-iron binding site; (ii.) a potentially redox-active tyrosine residue; and (iii.) the ability to dimerise and form an inter-protein heme binding pocket within electron tunnelling distance of the di-iron binding site. Upon replacing the heme with the photoactive zinc-chlorin e6 (ZnCe6) molecule and the di-iron binding site with two manganese ions, we show that the two Mn ions bind as a weakly coupled di-nuclear Mn2II,II centre, and that ZnCe6 binds in stoichiometric amounts of 1:2 with respect to the dimeric form of BFR. Upon illumination the bound ZnCe6 initiates electron transfer, followed by oxidation of the di-nuclear Mn centre possibly via one of the inherent tyrosine residues in the vicinity of the Mn cluster. The light dependent loss of the MnII EPR signals and the formation of low field parallel mode Mn EPR signals are attributed to the formation of MnIII species. The formation of the MnIII is concomitant with consumption of oxygen. Our model is the first artificial reaction centre developed for the photo-catalytic oxidation of a di-metal site within a protein matrix which potentially mimics water oxidation centre (WOC) photo-assembly.

Investigation of substrate water interactions at the high-affinity Mn site in the photosystem II oxygen-evolving complex.

18 O isotope exchange measurements of photosystem II (PSII) in thylakoids from wild-type and mutant Synechocystis have been performed to investigate binding of substrate water to the high-affinity Mn4 site in the oxygen-evolving complex (OEC). The mutants investigated were D1-D170H, a mutation of a direct ligand to the Mn4 ion, and D1-D61N, a mutation in the second coordination sphere. The substrate water 18 O exchange rates for D61N were found to be 0.16+/-0.02 s(-1) and 3.03+/-0.32 s(-1) for the slow and fast phases of exchange, respectively, compared with 0.47+/-0.04 s(-1) and 19.7+/-1.3 s(-1) for the wild-type. The D1-D170H rates were found to be 0.70+/-0.16 s(-1) and 24.4+/-4.6 s(-1) and thus are almost within the error limits for the wild-type rates. The results from the D1-D170H mutant indicate that the high-affinity Mn4 site does not directly bind to the substrate water molecule in slow exchange, but the binding of non-substrate water to this Mn ion cannot be excluded. The results from the D61N mutation show an interaction with both substrate water molecules, which could be an indication that D61 is involved in a hydrogen bonding network with the substrate water. Our results provide limitations as to where the two substrate water molecules bind in the OEC of PSII.

Glutamate-354 of the CP43 polypeptide interacts with the oxygen-evolving Mn4Ca cluster of photosystem II: a preliminary characterization of the Glu354Gln mutant.

In the recent X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is assigned as a ligand of the O2-evolving Mn4Ca cluster. In this communication, a preliminary characterization of the CP43-Glu354Gln mutant of the cyanobacterium Synechocystis sp. PCC 6803 is presented. The steady-state rate of O2 evolution in the mutant cells is only approximately 20% compared with the wild-type, but the kinetics of O2 release are essentially unchanged and the O2-flash yields show normal period-four oscillations, albeit with lower overall intensity. Purified PSII particles exhibit an essentially normal S2 state multiline electron paramagnetic resonance (EPR) signal, but exhibit a substantially altered S2-minus-S1 Fourier transform infrared (FTIR) difference spectrum. The intensities of the mutant EPR and FTIR difference spectra (above 75% compared with wild-type) are much greater than the O2 signals and suggest that CP43-Glu354Gln PSII reaction centres are heterogeneous, with a minority fraction able to evolve O2 with normal O2 release kinetics and a majority fraction unable to advance beyond the S2 or S3 states. The S2-minus-S1 FTIR difference spectrum of CP43-Glu354Gln PSII particles is altered in both the symmetric and asymmetric carboxylate stretching regions, implying either that CP43-Glu354 is exquisitely sensitive to the increased charge that develops on the Mn4Ca cluster during the S1-->S2 transition or that the CP43-Glu354Gln mutation changes the distribution of Mn(III) and Mn(IV) oxidation states within the Mn4Ca cluster in the S1 and/or S2 states.

Engineering model proteins for Photosystem II function.

Our knowledge of Photosystem II and the molecular mechanism of oxygen production are rapidly advancing. The time is now ripe to exploit this knowledge and use it as a blueprint for the development of light-driven catalysts, ultimately for the splitting of water into O2 and H2. In this article, we outline the background and our approach to this technological application through the reverse engineering of Photosystem II into model proteins.