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Abstract  In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein. [ABSTRACT FROM AUTHOR]

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., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

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.

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.

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., (© Springer Science+Business Media B.V. 2009)

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.

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.

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.

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein-protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.

Using a carbonic anhydrase assay based on membrane inlet mass spectrometry (MIMS), we have extended our earlier investigations of Photosystem II (PSII)-associated carbonic anhydrase activity in spinach PSII preparations (W. Hillier, I. McConnell, M. R. Badger, A. Boussac, V.V. Klimov G. C. Dismukes, T. Wydrzynski Biochemistry 2006, 45:2094). The relationship between the carbonic anhydrase activity and O(2) evolution has been evaluated in terms of the effects of metal ion addition, preparation type, light, and response to specific inhibitors. The results indicate that the PSII-associated carbonic anhydrase activity is variable and appears not to be associated specifically with the oxygen evolving activity nor the 33 kDa extrinsic manganese stabilising protein.

Much is now known about how protein folding occurs, through the sequence analysis of proteins of known folding geometry and the sequence/structural analysis of proteins and their mutants. This has allowed not only the modification of natural proteins but also the construction of de novo polypeptides with predictable folding patterns. Structure/function analysis of natural proteins is used to construct derived versions that retain a degree of biological activity. The constructed versions made of either natural or artificial sequences contain critical residues for activity such as receptor binding. In some cases, the functionality is introduced by incorporating binding sites for other elements, such as organic cofactors or transition metals, into the protein scaffold. While these modified proteins can mimic the function of natural proteins, they can also be constructed to have novel activities. Recently engineered photoactive proteins are good examples of such systems in which a light-induced electron transfer can be established in normally light-insensitive proteins. The present review covers some aspects of protein design that have been used to investigate protein receptor binding, cofactor binding and biological electron transfer.

In vitro chlorophyll (Chl) aggregates have often served as models for in vivo forms of long-wavelength Chl. However, the interaction of protein-bound Chl molecules is typically different than that occurring in solvent-based self-aggregates. We have chosen a water-soluble Chl-binding protein (WSCP) from cauliflower in order to help characterize the spectroscopic properties of Chl in a single well-defined native environment and also to study the pigment-pigment (exciton) interactions present in assemblies of this protein. WSCP forms tetrameric units upon binding two Chl molecules. We present the absorption, circular dichroism (CD), magnetic circular dichroism (MCD), and emission spectra at 1.7 K of recombinant WSCP tetramers containing either Chl a or Chl d. The spectroscopic characteristics provide evidence for significant exciton interaction between equivalent Chl molecules. Our simple exciton analysis allows an estimate of the molecular geometry of the dimer, which is predicted to have an "open sandwich"-type structure. We find that the ratio of the magnetic circular dichroism to absorption, deltaA/A, is substantially increased (approximately 60%) for Chl a in this system compared to its value in solution. This increase is in marked contrast to substantial reductions (>50%) of deltaA/A seen in solvent-based Chl aggregates and in photosynthetic reaction centers. Current theoretical models are unable to account for such large variations in the MCD to absorption ratio for Chl. We propose that spectroscopic studies of WSCP mutants will enable a fundamental understanding of Chl-Chl and Chl-protein interactions.

On the basis of equilibrium isotopic distribution experiments using (18)O-labeled water, it is generally accepted that water is the sole substrate for O(2) production by photosystem II (PSII). Nevertheless, recent studies indicating a direct interaction between bicarbonate and the donor side of PSII have been used to hypothesize that bicarbonate may have been a physiologically important substrate for O(2) production during the evolution of PSII [Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., and Tyryshikin, A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2170-2175]. To test out this hypothesis and to determine whether contemporary oxygenic organisms have the capacity to oxidize bicarbonate, we employed special rapid-mixing isotopic experiments using (18)O/(13)C-labeled bicarbonate to quantify the inherent carbonic anhydrase activity in PSII samples and the potential flux of oxygen from bicarbonate into the photosynthetically produced O(2). The measurements were made on PSII samples prepared from spinach, Thermosynechococcus elongatus, and Arthrospira maxima. For the latter organism, a strain was used that grows naturally in an alkaline, high (bi)carbonate soda lake in Africa. The results reveal that bicarbonate is not the substrate for O(2) production in these contemporary oxygenic photoautotrophs when assayed under single turnover conditions.

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.

The central photochemical reaction in photosystem II of green algae and plants and the reaction center of some photosynthetic bacteria involves a one-electron transfer from a light-activated chlorin complex to a bound quinone molecule. Through protein engineering, we have been able to modify a protein to mimic this reaction. A unique quinone-binding site was engineered into the Escherichia coli cytochrome b(562) by introducing a cysteine within the hydrophobic interior of the protein. Various quinones, such as p-benzoquinone and 2,3-dimethoxy-5-methyl-1,4-benzoquinone, were then covalently attached to the protein through a cysteine sulfur addition reaction to the quinone ring. The cysteine placement was designed to bind the quinone approximately 10 A from the edge of the bound porphyrin. Fluorescence measurements confirmed that the bound hydroquinone is incorporated toward the protein's hydrophobic interior and is partially solvent-shielded. The bound quinones remain redox-active and can be oxidized and rereduced in a two-electron process at neutral pH. The semiquinone can be generated at high pH by a one-electron reduction, and the midpoint potential of this can be adjusted by approximately 500 mV by binding different quinones to the protein. The heme-binding site of the modified cytochrome was then reconstituted with the chlorophyll analogue zinc chlorin e(6). By using EPR and fast optical techniques, we show that, in the various chlorin-protein-quinone complexes, light-induced electron transfer can occur from the chlorin to the bound oxidized quinone but not the hydroquinone, with electron transfer rates in the order of 10(8) s(-1).

Manganese (Mn) and calcium (Ca) are both metal cofactors required for photosynthetic oxygen evolution. The functional roles for these ions in the O2-evolving reactions are not completely known. They are studied by comparative spectroscopic measurements between intact and metal-depleted samples. In this chapter, we outline three experimental procedures used for the various removal of Mn and Ca from photosystem (PS) II-containing (i.e,. O2-evolving) preparations: the complete Mn extraction using a strong alkaline Ches buffer (pH 9.4)/MgCl2 wash, partial Mn extraction using a mild hydroxylamine (pH 6.8) wash, and specific Ca extraction through a low pH/citrate (pH 3) wash. The O2 evolution activities (measured by a Clarke-type oxygen electrode), protein composition (determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis), and the relative Mn and Ca content (measured by atomic absorption spectroscopy) are reported for each extraction procedure.

Direct evidence is presented to show that calcium is inherently involved in the binding of one of the two substrate-water molecules to the oxygen-evolving complex in photosystem II. Previous rapid (millisecond range) (18)O isotope exchange measurements between added H(2)(18)O and the photogenerated O(2) have shown that the two substrate-water molecules bind to separate sites throughout the S-state cycle, as revealed by their kinetically distinct rates of (18)O exchange [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399-4405]. Upon extraction of the functionally bound calcium using a either a low-pH/citrate treatment or a NaCl/A23187/EGTA treatment and subsequent reconstitution of activity with strontium, we show for the first time a specific increase in the slow rate of (18)O exchange by a factor of 3-4. This increase in the slow rate of exchange is consistently observed across the S(1), S(2), and S(3) states. In contrast, the fast phase of (18)O exchange in the S(3) state appears to be affected little upon strontium reconstitution, while the fast phases of exchange in the S(1) and S(2) states remain largely unresolvable, at the detectable limits of the current techniques. The results are discussed in terms of a possible substrate bridging structure between the functional calcium and a catalytic manganese ion that gives rise to the slowly exchanging component.

Net energy accumulation by marine microalgae at very low photon fluxes involves modulation of several attributes related to both the growth and photosynthetic physiology of these organisms. Here we studied flash-induced oscillatory patterns in oxygen evolution by previously dark-adapted cells of the green alga Dunaliella tertiolecta (Butcher) and the diatom Phaeodactylum tricornutum (Bohlin). The activity of the oxygen-evolving complex was found to be species-specific and influenced by photoacclimation. Results from measurements of oxygen flash yield obtained for these organisms grown under light-saturating conditions are directly comparable to those previously reported in the literature for other microalgae and higher plants. However, similar measurements on cells grown in low-light and/or light-starved conditions indicate an increased level of backward transitions (double misses) leading to the formation of super-reduced states (i.e. S -1 and S -2 ). Thus, in this communication, we present the first evidence that super-reduced states can be generated in vivo and speculate, on how they may be physiologically important.

We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.

The first direct evidence which shows that both substrate-water molecules are bound to the O(2)-evolving catalytic site in the S(2) state of photosystem II (PSII) is presented. Rapid (18)O isotope exchange measurements between H(2)(18)O incubated in the S(2) state of PSII-enriched membrane samples and the photogenerated O(2) reveal a fast and a slow phase of exchange at m/e 34 (which measures the level of the (16)O(18)O product). The rate constant for the slow phase of exchange ((34)k(1)) equals 1.9 +/- 0.3 s(-1) at 10 degrees C, while the fast phase of exchange is unresolved by our current experimental setup ((34)k(2) >or= 175 s(-1)). The unresolvable fast phase has left open the possibility that the second substrate-water molecule binds to the catalytic site only after the formation of the S(3) state [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399-4405]. However, for PSII samples depleted of the 17 and 23 kDa extrinsic proteins (Ex-depleted PSII), two completely resolvable phases of (18)O exchange are observed in the S(2) state of the residual activity, with the following rate constants: (34)k(1) = 2.6 +/- 0.3 s(-1) and (34)k(2) = 120 +/- 14 s(-1) at 10 degrees C. Upon addition of 15 mM CaCl(2) to Ex-depleted PSII, the O(2) evolution activity increases to approximately 80% of the control level, while the two resolvable phases of exchange remain the same. In measurements of Ex-depleted PSII at m/e 36 (which measures the level of the (18)O(18)O product), only a single phase of exchange is observed in the S(2) state, with a rate constant ((36)k(1) = 2.5 +/- 0.2 s(-1)) that is identical to the slow rate of exchange in the m/e 34 data. Taken together, these results show that the fast phase of (18)O exchange is specifically slowed by the removal of the 17 and 23 kDa extrinsic proteins and that the two substrate-water molecules must be bound to independent sites already in the S(2) state. In contrast, the (18)O exchange behavior in the S(1) state of Ex-depleted PSII is no different from what is observed for the control, with or without the addition of CaCl(2). Since the fast phase of exchange in the S(1) state is unresolved (i.e., (34)k(2) > 100 s(-1)), the possibility remains that the second substrate-water molecule binds to the catalytic site only after the formation of the S(2) state. The role of the 17 and 23 kDa extrinsic proteins in establishing an asymmetric dielectric environment around the substrate binding sites is discussed.

The Photosystem II (PSII) core antenna chlorophyll a-binding protein, CP47, contains six membrane-spanning alpha-helices separated by five hydrophilic loops: A-E. To identify important hydrophilic cytosolic regions, oligonucleotide-directed mutagenesis was employed to introduce short segment deletions into loops B and D, and the C-terminal domain. Four strains carrying deletions of between three and five residues were created in loop B. Two strains, with deletions adjacent to helices II and III, did not assemble PSII; however, the mutants delta(F123-D125) and delta(R127-S131) remained photoautotrophic with near wild-type levels of assembled reaction centers. In contrast, all deletions introduced into loop D, connecting helices IV and V, failed to assemble significant levels of PSII and were obligate photoheterotrophic mutants. However, deletions in the C-terminal domain did not prevent the assembly of PSII reaction centers although the mutant delta(S471 -T473), with a deletion adjacent to helix V1, exhibited retarded Q(A)- oxidation kinetics and the PSII-specific herbicide, atrazine, bound less tightly in the delta(S471-T473) and delta(F475-D477) strains. Deletions in the C-terminal domain also created mutants with large protein aggregates that were recognized by an antibody raised against the PSII reaction center D1 protein. Low-temperature fluorescence emission spectra of photoautotrophic strains carrying deletions in either the C-terminal domain or loop B did not provide evidence for impaired energy transfer from the phycobilisomes to the PSII reaction center. The data therefore suggest an important structural role for loop D in the assembly of PSII and a potential interaction between the C-terminal domain of CP47 and the PSII reaction center that, when perturbed, results in photoinduced protein aggregates involving the D1 protein.

Preparation of a minimum PSII core complex from spinach is described, containing four Mn per reaction center (RC) and exhibiting high O2 evolving activity [approximately 4000 micromol of O2 (mg of chl)(-1) x h(-1)]. The complex consists of the CP47 and CP43 chlorophyll binding proteins, the RC D1/D2 pair, the cytochrome b559 subunits, and the Mn-stabilizing psbO (33 kDa) protein, all present in the same stoichiometric amounts found in the parent PSII membranes. Several small subunits are also present. The cyt b559 content is 1.0 per RC in core complexes and PSII membranes. The total chlorophyll content is 32 chl a and <1 chl b per RC, the lowest yet reported for any active PSII preparation. The core complex exhibits the characteristic EPR signals seen in the S2 state of higher plant PSII. A procedure for preparing low-temperature samples of very high optical quality is developed, allowing detailed optical studies in the S1 and S2 states of the system to be made. Optical absorption, CD, and MCD spectra reveal unprecedented detail, including a prominent, well-resolved feature at 683.5 nm (14630 cm(-1)) with a weaker partner at 187 cm(-1) to higher energy. On the basis of band intensity, CD, and MCD arguments, these features are identified as the exciton split components of P680 in an intact, active reaction center special pair. Comparisons are made with solubilized D1/D2/cyt b559 material and cyanobacterial PSII.

The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state. However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.

The mechanism for photosynthetic O2 evolution by photosystem II is currently a topic of intense debate. Important questions remain as to what is the nature of the binding sites for the substrate water and how does the O-O bond form. Recent measurements of the 18O exchange between the solvent water and the photogenerated O2 as a function of the S-state cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399-4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S0 state, while the second substrate water molecule binds in the S3 state or possibly earlier. It may be that the second substrate water molecule only enters the catalytic sequence following the formation of the S3 state. Most importantly, comparison of the observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water molecules are most likely bound to separate Mn(III) ions, which do not undergo metal-centered oxidations through to the S3 state. The implication of this analysis is that in the S1 state, all four Mn ions are in the +3 oxidation state. This minireview summarizes the arguments for this proposal.

The first determinations of substrate water binding to the O(2) evolving complex in photosystem II as a complete function of the S states have been made. H(2)(18)O was rapidly injected into spinach thylakoid samples preset in either the S(0), S(1), S(2), or S(3) states, and the rate of (18)O incorporation into the O(2) produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the (16)O(18)O product), the rate of (18)O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 degrees C of 8 +/- 2, 0.021 +/- 0.002, 2.2 +/- 0.3, and 1.9 +/- 0.2 s(-1) for the S(0), S(1), S(2), and S(3) states, respectively, while the fast phase kinetics yield a rate constant of 36.8 +/- 1.9 s(-1) for the S(3) state but remain unresolvable (>100 (s-1)) for the S(0), S(1), and S(2) states. Comparisons of the (18)O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S(0) to S(1) transition, then decreases during the S(1) to S(2) transition, but stays the same during the S(2) to S(3) transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S(2) to S(3) transition. These findings are discussed in terms of two independent Mn(III) substrate binding sites within the O(2) evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn(III) sites may only first bind a substrate water molecule during the S(2) to S(3) transition.

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.

Time-resolved FTIR spectroscopy has been used to kinetically characterize the vibrational properties of intact photosystem II-enriched membrane samples undergoing the S1QB-to-S2QB- transition at room temperature. To optimize the experimental conditions for the FTIR measurements, oxygen polarographic and variable chlorophyll a fluorescence measurements were used to define the decay of S2 and QA-, respectively. The flash-induced S2QB-/S1QB difference spectra were measured at a temporal resolution of 4.44 s and a spectral resolution of 4 cm-1. An intense positive band is observed at 1480 cm-1 in the difference spectrum and shows a slow decay with a half time of approximately 13 s. Based on its decay kinetics and analogy to the infrared absorption of QA- of photosystem II and QB- in bacterial reaction centers, we conclude that the 1480 cm-1 band arises from QB- of PSII and tentatively assign it to the upsilon(CO) mode of the semiquinone anion QB-. The infrared spectral features attributed to the S1-to-S2 transition of the Mn cluster at room temperature show striking similarity to the S2/S1 difference spectrum measured at cryogenic temperatures (Noguchi, T., Ono, T.-A., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200).

The reaction center-binding protein D1 of photosystem II (PS II) undergoes rapid turnover under light stress conditions. In the present study, we investigated the role of the extrinsic 33 kDa protein (OEC33) in the early stages of D1 turnover. D1 degradation was measured after strong illumination (1000-5000 microE m-2 S-1) of spinach manganese-depleted, PSII-enriched membrane and core samples in the presence and absence of the OEC33 under aerobic conditions at room temperature. PSII samples lacking the OEC33 were prepared by standard biochemical treatments with Tris or CaCl2/NH2OH while samples retaining the OEC33 were prepared with NH2OH or NaCl/NH2OH. The degradation of D1, monitored by SDS/urea-polyacrylamide gel electrophoresis and Western blotting using specific antibodies against D1, proceeds to a greater extent in NH2OH-treated samples than in Tris-treated samples over a 60 min illumination period. Under the same conditions, significantly more aggregation of D1 occurs in the Tris-treated samples than in the NH2OH-treated samples. The lower level of D1 degradation in Tris-treated samples is not due to secondary proteolysis, as judged from the time course for degradation at 25 degrees C or the degradation pattern at 4 degrees C. Similarly, for NaCl/NH2OH-treated samples, D1 degradation is greater and D1 aggregation less than in CaCl2/NH2OH-treated samples. The effect of the presence of the OEC33 on D1 degradation and aggregation is confirmed by reconstitution experiments in which the isolated OEC33 is restored back to Tris-treated samples. During very strong illumination, significant loss of CP43 also occurs in Tris-treated but not in NH2OH-treated samples. Structural analysis of PS II core complexes by Fourier transform infrared (FT-IR) spectroscopy revealed very little change in the protein secondary structure after 10 min illumination of NH2OH-treated samples while a large 10% decrease of alpha-helix content occurs in Tris-treated samples. On the basis of these results, we suggest that either (1) the OEC33 stabilizes the structural integrity of PS II such that it prevents the photodamaged D1 protein from aggregating with nearby polypeptides and thereby facilitating degradation or (2) the OEC33 specifically stabilizes CP43, a putative D1-specific protease, which normally promotes the efficient degradation of D1.

Decay of Signal IIvf of photosystem II (PSII), under repetitive flash conditions, was examined in whole cells of wild-type Synechocystis sp. PCC6803 and in cells of an engineered strain, delta psbO, which lacks the extrinsic 33 kDa manganese-stabilizing protein (MSP). Previous polarographic analysis had shown that O2 release during the S3-->[S4]-->S0 transition of the catalytic cycle is significantly retarded in the delta psbO strain relative to the wild-type [Burnap et al. (1992) Biochemistry 31, 7404-7410]. The present experiments provide evidence that a parallel retardation in the rate of reduction of photooxidized Yz by the H2O oxidation complex is due to the absence of MSP. The half-time of the Signal IIvf component, corresponding to Yz. reduction during the S3-->[S4]-->S0 transition, was estimated to be 1.2 and 6.0 ms in the wild-type and delta psbO cells, respectively.

In this paper, we present the first time-dependent measurements of flash-induced infrared difference spectra of photosystem II (PSII) using Fourier transform infrared (FTIR) spectroscopy. With this experimental approach, we were able to obtain the YZoxQA-/YZQA vibrational difference spectrum of Tris-washed, PSII-enriched samples in the absence of hydroxylamine at room temperature (16 +/- 2 degrees C), with a spectral resolution of 4 cm-1 and a temporal resolution of 50 ms. In order to determine the dominant species in the FTIR spectrum at a particular point in time after an excitation flash, the decay kinetics of YZox and QA- were independently monitored by EPR and chlorophyll a fluorescence, respectively, under the same experimental conditions. These measurements confirmed that the addition of DCMU to Tris-washed PSII samples does not significantly affect the YZox decay, but does substantially slow down the QA- decay. By making use of the difference in the decay kinetics using DCMU, the QA-/QA signals could be separated from the YZox/YZ signals and a pure QA-/QA difference spectrum obtained. By comparison of the YZoxQA-/YZQA difference spectrum with the pure QA-/QA difference spectrum, a large differential band at 1706/1699 cm-1 could be identified and associated with YZ oxidation. In contrast, an intense band at 1478 cm-1, whose DCMU-sensitive decay follows the QA- decay based on the chlorophyll a fluorescence measurements, was present in all of the time-resolved spectra. Since no significant reversible Chl+ radicals could be detected by the EPR measurements under our experimental conditions, we confirm that this band most likely arises only from the semiquinone anion QA- [Berthomieu, C., Nabedryk, E., Mäntele, W., & Breton, J. (1990) FEBS Lett. 269, 363-367].

The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.