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 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.

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.

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.

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.

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.

Creating functional artificial proteins.

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.

Magneto-optic spectroscopy of a protein tetramer binding two exciton-coupled chlorophylls.

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.

Quantitative assessment of intrinsic carbonic anhydrase activity and the capacity for bicarbonate oxidation in photosystem II.

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.

Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina.

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.

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron.

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.

Protein engineering of cytochrome b562 for quinone binding and light-induced electron transfer.

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).

Extraction of the functional manganese and calcium from photosystem II.

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.

(18)O isotope exchange measurements reveal that calcium is involved in the binding of one substrate-water molecule to the oxygen-evolving complex in photosystem II.

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.

Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation.

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.

Photoacclimation involves modulation of the photosynthetic oxygen-evolving reactions in Dunaliella tertiolecta and Phaeodactylum tricornutum.

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.

Amino acid deletions in the cytosolic domains of the chlorophyll a-binding protein CP47 slow Q(A)- oxidation and/or prevent the assembly of photosystem II.

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.

The two substrate-water molecules are already bound to the oxygen-evolving complex in the S2 state of photosystem II.

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.

Magneto-optical measurements of the pigments in fully active photosystem II core complexes from plants.

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.