The nonheme iron in photosystem II

Photosystem II (PSII), the light-driven water:plastoquinone (PQ) oxidoreductase of oxygenic photosynthesis, contains a nonheme iron (NHI) at its electron acceptor side. The NHI is situated between the two PQs Q_A and Q_B that serve as one-electron transmitter and substrate of the reductase part of PSII, respectively. Among the ligands of the NHI is a (bi)carbonate originating from CO₂, the substrate of the dark reactions of oxygenic photosynthesis. Based on recent advances in the crystallography of PSII, we review the structure of the NHI in PSII and discuss ideas concerning its function and the role of bicarbonate along with a comparison to the reaction center of purple bacteria and other enzymes containing a mononuclear NHI site.     

Decoupling of the processes of molecular oxygen synthesis and electron transport in Ca2+-depleted PSII membranes

Extraction of Ca(2+) from the O(2)-evolving complex (OEC) of photosystem II (PSII) membranes with 2 M NaCl in the light (PSII(-Ca/NaCl)) results in 90% inhibition of the O(2)-evolution reaction. However, electron transfer from the donor to acceptor side of PSII, measured as the reduction of the exogenous acceptor 2,6-dichlorophenolindophenol (DCIP) under continuous light, is inhibited by only 30%. Thus, calcium extraction from the OEC inhibits the synthesis of molecular O(2) but not the oxidation of a substrate we term X, the source of electrons for DCIP reduction. The presence of electron transfer across PSII(-Ca/NaCl) membranes was demonstrated using fluorescence induction kinetics, a method that does not require an artificial acceptor. The calcium chelator, EGTA (5 mM), when added to PSII(-Ca/NaCl) membranes, does not affect the inhibition of O(2) evolution by NaCl but does inhibit DCIP reduction up to 92% (the reason why electron transport in Ca(2+)-depleted materials has not been noticed before). Another chelator, sodium citrate (citrate/low pH method of calcium extraction), also inhibits both O(2) evolution and DCIP reduction. The role of all buffer components (including bicarbonate and sucrose) as possible sources of electrons for PSII(-Ca/NaCl) membranes was investigated, but only the absence of chloride anions strongly inhibited the rate of DCIP reduction. Substitution of other anions for chloride indicates that Cl(-) serves its well-known role as an OEC cofactor, but it is not substrate X. Multiple turnover flash experiments have shown a period of four oscillations of the fluorescence yield (both the maximum level, F(max), and the fluorescence level measured 50 s after an actinic flash in the presence of DCMU) in native PSII membranes, reflecting the normal function of the OEC, but the absence of oscillations in PSII(-Ca/NaCl) samples. Thus, PSII(-Ca/NaCl) samples do not evolve O(2) but do transfer electrons from the donor to acceptor sides and exhibit a disrupted S-state cycle. We explain these results as follows. In Ca(2+)-depleted PSII membranes, obtained without chelators, the oxidation of the OEC stops after the absorption of three quanta of light (from the S1 state), which should convert the native OEC to the S4 state. An one-electron oxidation of the water molecule bound to the Mn cluster then occurs (the second substrate water molecule is absent due to the absence of calcium), and the OEC returns to the S3 state. The appearance of a sub-cycle within the S-state cycle between S3-like and S4-like states supplies electrons (substrate X is postulated to be OH(-)), explains the absence of O(2) production, and results in the absence of a period of four oscillation of the normal functional parameters, such as the fluorescence yield or the EPR signal from S2. Chloride anions probably keep the redox potential of the Mn cluster low enough for its oxidation by Y(Z)(*).     

Identification of a Mn-O-Mn cluster vibrational mode of the oxygen-evolving complex in photosystem II by low-frequency FTIR spectroscopy

We have developed conditions for recording the low-frequency S(2)/S(1) Fourier transform infrared difference spectrum of hydrated PSII samples. By exchanging PSII samples with buffered (18)O water, we found that a positive band at 606 cm(-)(1) in the S(2)/S(1) spectrum in (16)O water is clearly downshifted to 596 cm(-)(1) in (18)O water. By taking double-difference (S(2)/S(1) and (16)O minus (18)O) spectra, we assign the 606 cm(-)(1) mode to an S(2) mode and also identify a corresponding S(1) mode at about 625 cm(-)(1). In addition, by Sr and (44)Ca substitution experiments, we found that the 606 cm(-)(1) mode is upshifted to about 618 cm(-)(1) by Sr(2+) substitution but that this mode is not affected by substitution with the (44)Ca isotope. On the basis of these results and also on the basis of studies of Mn model compounds, we assign the 625 cm(-)(1) mode in the S(1) state and the 606 cm(-)(1) mode in the S(2) state to a Mn-O-Mn cluster vibration of the oxygen-evolving complex (OEC) in PSII. This structure may include additional bridge(s), which could be another oxo, carboxylato(s), or atoms derived from an amino acid side chain. Our results indicate that the bridged oxygen atom shown in this Mn-O-Mn cluster is exchangeable and accessible by water. The downshift in the Mn-O-Mn cluster vibration as manganese is oxidized during the S(1) --> S(2) transition is counterintuitive; we discuss possible origins of this behavior. Our results also indicate that Sr(2+) substitution in PSII causes a small structural perturbation that affects the bond strength of the Mn-O-Mn cluster in the PSII OEC. This suggests that Sr(2+), and by inference, Ca(2+), communicates with, but is not integral to, the manganese core.     

Mutagenesis of CP43-arginine-357 to serine reveals new evidence for (bi)carbonate functioning in the water oxidizing complex of Photosystem II.

The chlorophyll-binding protein CP43 is an inner subunit of the Photosystem II (PSII) reaction center core complex of all oxygenic photoautotrophs. X-Ray structural evidence places the guanidinium cation of the conserved arginine 357 residue of CP43 within a few Angstroms to the Mn(4)Ca cluster of the water-oxidizing complex (WOC) and has been implicated as a possible carbonate binding site. To test the hypothesis, the serine mutant, CP43-R357S, from Synechocystis PCC 6803 was investigated by PSII variable fluorescence (F(v)/F(m)) and simultaneous flash O(2) yield measurements in cells and thylakoid membranes. The R357S mutant assembles PSII-WOC centers, but is unable to grow photoautotrophically. Reconstitution of O(2) evolution by photoactivation and the occurrence of period-four oscillations of F(v)/F(m) establishes that the R357S mutant contains an assembled Mn(4)Ca cluster, but turnover is impaired as seen by an 11-fold larger Kok double miss parameter and faster decay of upper S states. Using pulsed light to avoid photoinactivation, wild-type cells and thylakoid membranes exhibit a 2-4-fold loss in O(2) evolution rate upon partial bicarbonate depletion under multiple turnover conditions, while the R357S mutant is unaffected by bicarbonate. Arginine R357 appears to function in binding a (bi)carbonate ion essential to normal catalytic turnover of the WOC. The quantum yield of electron donation from the WOC into PSII increases with decreasing turnover rate in R357S mutant cells and involves an aborted two-flash pathway that is distinct from the classical four-flash pattern. We speculate that an altered photochemical mechanism for O(2) production occurs via formation of hydrogen peroxide, by analogy to other treatments that retard the kinetics of proton release into the lumen.     

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.     

Photosystem II and the unique role of bicarbonate: A historical perspective

In photosynthesis, cyanobacteria, algae and plants fix carbon dioxide (CO(2)) into carbohydrates; this is necessary to support life on Earth. Over 50years ago, Otto Heinrich Warburg discovered a unique stimulatory role of CO(2) in the Hill reaction (i.e., O(2) evolution accompanied by reduction of an artificial electron acceptor), which, obviously, does not include any carbon fixation pathway; Warburg used this discovery to support his idea that O(2) in photosynthesis originates in CO(2). During the 1960s, a large number of researchers attempted to decipher this unique phenomenon, with limited success. In the 1970s, Alan Stemler, in Govindjee's lab, perfected methods to get highly reproducible results, and observed, among other things, that the turnover of Photosystem II (PSII) was stimulated by bicarbonate ions (hydrogen carbonate): the effect would be on the donor or the acceptor, or both sides of PSII. In 1975, Thomas Wydrzynski, also in Govindjee's lab, discovered that there was a definite bicarbonate effect on the electron acceptor (the plastoquinone) side of PSII. The most recent 1.9? crystal structure of PSII, unequivocally shows HCO(3)(-) bound to the non-heme iron that sits in-between the bound primary quinone electron acceptor, Q(A), and the secondary quinone electron acceptor Q(B). In this review, we focus on the historical development of our understanding of this unique bicarbonate effect on the electron acceptor side of PSII, and its mechanism as obtained by biochemical, biophysical and molecular biological approaches in many laboratories around the World. We suggest an atomic level model in which HCO(3)(-)/CO(3)(2-) plays a key role in the protonation of the reduced Q(B). In addition, we make comments on the role of bicarbonate on the donor side of PSII, as has been extensively studied in the labs of Alan Stemler (USA) and Vyacheslav Klimov (Russia). We end this review by discussing the uniqueness of bicarbonate's role in oxygenic photosynthesis and its role in the evolutionary development of O(2)-evolving PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution

It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.     

What Are the Oxidation States of Manganese Required To Catalyze Photosynthetic Water Oxidation?

Photosynthetic O(2) production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S(0) state to be (Mn(III))(3)Mn(IV). We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn(4)Ca core of WOC during photoassembly, starting from free Mn(II) and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O(2) molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S(0), to be three, indicating a net oxidation state three equivalents above four Mn(II), formally (Mn(III))(3)Mn(II), whereas the O(2) releasing state, S(4), corresponds formally to (Mn(IV))(3)Mn(III). The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation.     

Monitoring water reactions during the S-state cycle of the photosynthetic water-oxidizing center: detection of the DOD bending vibrations by means of Fourier transform infrared spectroscopy

Photosynthetic water oxidation takes place in the water-oxidizing center (WOC) of photosystem II (PSII). To clarify the mechanism of water oxidation, detecting water molecules in the WOC and monitoring their reactions at the molecular level are essential. In this study, we have for the first time detected the DOD bending vibrations of functional D 2O molecules during the S-state cycle of the WOC by means of Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra upon S-state transitions were measured using the PSII core complexes from Thermosynechococcus elongatus moderately deuterated with D 2 (16)O and D 2 (18)O. D 2 (16)O-minus-D 2 (18)O double difference spectra at individual S-state transitions exhibited six to eight peaks arising from the D (16)OD/D (18)OD bending vibrations in the 1250-1150 cm (-1) region. This observation indicates that at least two water molecules, not in any deprotonated forms, participate in the reaction at each S-state transition throughout the cycle. Most of the peaks exhibited clear counter peaks with opposite signs at different transitions, reflecting a series of reactions of water molecules at the catalytic site. In contrast, negative bands at approximately 1240 cm (-1) in the S 2 --> S 3, S 3 --> S 0, and possibly S 0 --> S 1 transitions, for which no clear counter peaks were found in other transitions, can be interpreted as insertion of substrate water into the WOC from a water cluster in the proteins. The characteristics of the weakly D-bonded OD stretching bands were consistent with the insertion of substrate from internal water molecules in the S 2 --> S 3 and S 3 --> S 0 transitions. The results of this study show that FTIR detection of the DOD bending vibrations is a powerful method for investigating the molecular mechanism of photosynthetic water oxidation as well as other enzymatic reactions involving functional water molecules.     

Interaction between tyrosineZ and substrate water in active photosystem II

In the field of photosynthetic water oxidation it has been under debate whether Tyrosine(Z) (Tyr(Z)) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of Tyr(Z) with substrate water has been assumed. However, there is no direct evidence for the interaction between Tyr(Z) and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of Tyr(Z) oxidation at cryogenic temperature in the S(0) and S(1) states was around neutral pH and was essentially pH-independent. The yield of Tyr(Z) oxidation decreased at acidic and alkaline pH, with pKs at 4.7-4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of Tyr(Z)(.) in the S(0) and S(1) states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the Tyr(Z) oxidation and Tyr(Z)(.) reduction at cryogenic temperature in the S(0) and S(1) states of the active PSII. Theoretical calculations indicate that Tyr(Z) becomes more difficult to oxidize when a H(2)O molecule interacts directly with it. It is suggested that Tyr(Z) is probably located in a hydrophobic environment with no direct interaction with the substrate H(2)O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.     

The affinities for the two substrate water binding sites in the O(2) evolving complex of photosystem II vary independently during S-state turnover

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.     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Effects of methanol on the S i -state transitions in photosynthetic water-splitting

From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at-or at least very close to-the Mn(4)O(x)Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O(2) evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S( i ) state advancement on the methanol concentrations in the range of 0-10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn(4)O(x)Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S(4) --> S(0) and the other during the S(2) --> S(3) transition.     

Bicarbonate requirement for the water-oxidizing complex of photosystem II

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.     

Mono-manganese mechanism of the photosystem II water splitting reaction by a unique Mn4Ca cluster

The molecular mechanism of the water oxidation reaction in photosystem II (PSII) of green plants remains a great mystery in life science. This reaction is known to take place in the oxygen evolving complex (OEC) incorporating four manganese, one calcium and one chloride cofactors, that is light-driven to cycle four intermediates, designated S(0) through S(4), to produce four protons, five electrons and lastly one molecular oxygen, for indispensable resources in biosphere. Recent advancements of X-ray crystallography models established the existence of a catalytic Mn(4)Ca cluster ligated by seven protein amino acids, but its functional structure is not yet resolved. The (18)O exchange rates of two substrate water molecules were recently measured for four S(i)-state samples (i=0-3) leading to (34)O(2) and (36)O(2) formations, revealing asymmetric substrate binding sites significantly depending on the S(i)-state. In this paper, we present a chemically complete model for the Mn(4)Ca cluster and its surrounding enzyme field, which we found out from some possible models by using the hybrid density functional theoretic geometry optimization method to confirm good agreements with the 3.0 A resolution PSII model [B. Loll, J. Kern, W. Saenger, A. Zouni , J. Biesiadka, Nature 438 (2005) 1040-1044] and the S-state dependence of (18)O exchange rates [W. Hillier and T. Wydrzynski, Phys. Chem. Chem. Phys. 6 (2004) 4882-4889]. Furthermore, we have verified that two substrate water molecules are bound to asymmetric cis-positions on the terminal Mn ion being triply bridged (mu-oxo, mu-carboxylato, and a hydrogen bond) to the Mn(3)CaO(3)(OH) core, by developing a generalized theory of (18)O exchange kinetics in OEC to obtain an experimental evidence for the cross exchange pathway from the slow to the fast exchange process. Some important experimental data will be discussed in terms of this model and its possible tautomers, to suggest that a cofactor, Cl(-) ion, may be bound to CP43-Arg357 nearby Ca(2+) ion and that D1-His337 may be used to trap a released proton only in the S(2)-state.     

Calcium ligation in photosystem II under inhibiting conditions

In oxygenic photosynthesis, PSII carries out the oxidation of water and reduction of plastoquinone. The product of water oxidation is molecular oxygen. The water splitting complex is located on the lumenal side of the PSII reaction center and contains manganese, calcium, and chloride. Four sequential photooxidation reactions are required to generate oxygen from water; the five sequentially oxidized forms of the water splitting complex are known as the Sn states, where n refers to the number of oxidizing equivalents stored. Calcium plays a role in water oxidation; removal of calcium is associated with an inhibition of the S state cycle. Although calcium can be replaced by other cations in vitro, only strontium maintains activity, and the steady-state rate of oxygen evolution is decreased in strontium-reconstituted PSII. In this article, we study the role of calcium in PSII that is limited in water content. We report that strontium substitution or 18OH2 exchange causes conformational changes in the calcium ligation shell. The conformational change is detected because of a perturbation to calcium ligation during the S1 to S2 and S2 to S3 transition under water-limited conditions.     

An activated glutamate residue identified in photosystem II at the interface between the manganese-stabilizing subunit and the D2 polypeptide

Photosystem II (PSII) catalyzes the oxidation of water during oxygenic photosynthesis. PSII is composed both of intrinsic subunits, such as D1, D2, and CP47, and extrinsic subunits, such as the manganese-stabilizing subunit (MSP). Previous work has shown that amines covalently bind to amino acid residues in the CP47, D1, and D2 subunits of plant and cyanobacterial PSII, and that these covalent reactions are prevented by the addition of chloride in plant preparations depleted of the 18- and 24-kDa extrinsic subunits. It has been proposed that these reactive groups are carbonyl-containing, post-translationally modified amino acid side chains (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204-2209 and Anderson, L. B., Ouellette, A. J. A., and Barry, B. A. (2000) J. Biol. Chem. 275, 4920-4927). To identify the amino acid binding site in the spinach D2 subunit, we have employed a biotin-amine labeling reagent, which can be used in conjunction with avidin affinity chromatography to purify biotinylated peptides from the PSII complex. Multidimensional chromato-graphic separation and multistage mass spectrometry localizes a novel post-translational modification in the D2 subunit to glutamate 303. We propose that this glutamate is activated for amine reaction by post-translational modification. Because the modified glutamate is located at a contact site between the D2 and manganese-stabilizing subunits, we suggest that the modification is important in vivo in stabilizing the interaction between these two PSII subunits. Consistent with this conclusion, mutations at the modified glutamate alter the steady-state rate of photosynthetic oxygen evolution.     

Biochemical and morphological characterization of sulfur-deprived and H2-producing Chlamydomonas reinhardtii (green alga)

Sulfur deprivation in green algae causes reversible inhibition of photosynthetic activity. In the absence of S, rates of photosynthetic O2 evolution drop below those of O2 consumption by respiration. As a consequence, sealed cultures of the green alga Chlamydomonas reinhardtii become anaerobic in the light, induce the "Fe-hydrogenase" pathway of electron transport and photosynthetically produce H2 gas. In the course of such H2-gas production cells consume substantial amounts of internal starch and protein. Such catabolic reactions may sustain, directly or in directly, the H2-production process. Profile analysis of selected photosynthetic proteins showed a precipitous decline in the amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a function of time in S deprivation, a more gradual decline in the level of photosystem (PS) II and PSI proteins, and a change in the composition of the PSII light-harvesting complex (LHC-II). An increase in the level of the enzyme Fe-hydrogenase was noted during the initial stages of S deprivation (0-72 h) followed by a decline in the level of this enzyme during longer (t >72 h) S-deprivation times. Microscopic observations showed distinct morphological changes in C. reinhardtii during S deprivation and H2 production. Ellipsoid-shaped cells (normal photosynthesis) gave way to larger and spherical cell shapes in the initial stages of S deprivation and H2 production, followed by cell mass reductions after longer S-deprivation and H2-production times. It is suggested that, under S-deprivation conditions, electrons derived from a residual PSII H2O-oxidation activity feed into the hydrogenase pathway, thereby contributing to the H2-production process in Chlamydomonas reinhardtii. Interplay between oxygenic photosynthesis, mitochondrial respiration, catabolism of endogenous substrate, and electron transport via the hydrogenase pathway is essential for this light-mediated H2-production process.     

Effects of ammonia on the structure of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

NH(3) is a structural analogue of substrate H(2)O and an inhibitor to the water oxidation reaction in photosystem II. To test whether or not NH(3) is able to replace substrate water molecules on the oxygen-evolving complex in photosystem II, we studied the effects of NH(3) on the high-frequency region (3750-3550 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (pH 7.5 at 250 K), where OH stretch modes of weak hydrogen-bonded active water molecules occur. Our results showed that NH(3) did not replace the active water molecule on the oxygen-evolving complex that gave rise to the S(1) mode at ~3586 cm(-1) and the S(2) mode at ~3613 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. In addition, our mid-frequency FTIR results showed a clear difference between pH 6.5 and 7.5 on the concentration dependence of the NH(4)Cl-induced upshift of the S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectra of NH(4)Cl-treated PSII samples. Our results provided strong evidence that NH(3) induced this upshift in the spectra of NH(4)Cl-treated PSII samples at 250 K. Moreover, our low-frequency FTIR results showed that the Mn-O-Mn cluster vibrational mode at 606 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the NaCl control PSII sample was diminished in those samples treated with NH(4)Cl. Our results suggest that NH(3) induced a significant alteration on the core structure of the Mn(4)CaO(5) cluster in PSII. The implication of our findings on the structure of the NH(3)-binding site on the OEC in PSII will be discussed.     

Low-frequency fourier transform infrared spectroscopy of the oxygen-evolving and quinone acceptor complexes in photosystem II

The low-frequency (<1000 cm-1) region of the IR spectrum has the potential to provide detailed structural and mechanistic insight into the photosystem II/oxygen evolving complex (PSII/OEC). A cluster of four manganese ions forms the core of the OEC and diagnostic manganese-ligand and manganese-substrate modes are expected to occur in the 200-900 cm-1 range. However, water also absorbs IR strongly in this region, which has limited previous Fourier transform infrared (FTIR) spectroscopic studies of the OEC to higher frequencies (>1000 cm-1). We have overcome the technical obstacles that have blocked FTIR access to low-frequency substrate, cofactor, and protein vibrational modes by using partially dehydrated samples, appropriate window materials, a wide-range MCT detector, a novel band-pass filter, and a closely regulated temperature control system. With this design, we studied PSII/OEC samples that were prepared by brief illumination of O2 evolving and Tris-washed preparations at 200 K or by a single saturating laser flash applied to O2 evolving and inhibited samples at 250 K. These protocols allowed us to isolate low-frequency modes that are specific to the QA-/QA and S2/S1 states. The high-frequency FTIR spectra recorded for these samples and parallel EPR experiments confirmed the states accessed by the trapping procedures we used. In the S2/S1 spectrum, we detect positive bands at 631 and 602 cm-1 and negative bands at 850, 679, 664, and 650 cm-1 that are specifically associated with these two S states. The possible origins of these IR bands are discussed. For the low-frequency QA-/QA difference spectrum, several modes can be assigned to ring stretching and bending modes from the neutral and anion radical states of the quinone acceptor. These results provide insight into the PSII/OEC and demonstrate the utility of FTIR techniques in accessing low-frequency modes in proteins.