S-state dependent Fourier transform infrared difference spectra for the photosystem II oxygen evolving complex

Vibrational spectroscopy provides a means to investigate molecular interactions within the active site of an enzyme. We have applied difference FTIR spectroscopy coupled with a flash turnover protocol of photosystem II (PSII) to study the oxygen evolving complex (OEC). Our data show two overlapping oscillatory patterns as the sample is flashed through the four-step S-state cycle that produces O(2) from two H(2)O molecules. The first oscillation pattern of the spectra shows a four-flash period four oscillation and reveals a number of new vibrational modes for each S-state transition, indicative of unique structural changes involved in the formation of each S-state. Importantly, the first and second flash difference spectra are reproduced in the 1800-1200 cm(-)(1) spectral region by the fifth and sixth flash difference spectra, respectively. The second oscillation pattern observed is a four-flash, period-two oscillation associated with changes primarily to the amide I and II modes and reports on changes in sign of these modes that alternate 0:0:1:1 during S-state advance. This four-flash, period-two oscillation undergoes sign inversion that alternates during the S(1)-to-S(2) and S(3)-to-S(0) transitions. Underlying this four-flash period two is a small-scale change in protein secondary structure in the PSII complex that is directly related to S-state advance. These oscillation patterns and their relationships with other PSII phenomena are discussed, and future work can initiate more detailed vibrational FTIR studies for the S-state transitions providing spectral assignments and further structural and mechanistic insight into the photosynthetic water oxidation reaction.     

Structural perturbation of the carboxylate ligands to the manganese cluster upon Ca2+/Sr2+ exchange in the S-state cycle of photosynthetic oxygen evolution as studied by flash-induced FTIR difference spectroscopy

A Ca(2+) ion is an indispensable element in the oxygen-evolving Mn cluster in photosystem II (PSII). To investigate the structural relevance of Ca(2+) to the Mn cluster, the effects of Sr(2+) substitution for Ca(2+) on the structures and reactions of ligands to the Mn cluster during the S-state cycle were investigated using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectra representing the four S-state transitions, S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1), were recorded by applying four consecutive flashes either to PSII core complexes from Thermosynechococcus elongatus or to PSII-enriched membranes from spinach. The spectra were also recorded using biosynthetically Sr(2+)-substituted PSII core complexes from T. elongatus and biochemically Sr(2+)-substituted PSII membranes from spinach. Several common spectral changes upon Sr(2+) substitution were observed in the COO(-) stretching region of the flash-induced spectra for both preparations, which were best expressed in Ca(2+)-minus-Sr(2+) double difference spectra. The significant intensity changes in the symmetric COO(-) peaks at approximately 1364 and approximately 1418 cm(-)(1) at the first flash were reversed as opposite intensity changes at the third flash, and the slight shift of the approximately 1446 cm(-)(1) peak at the second flash corresponded to the similar but opposite shift at the fourth flash. Analyses of these changes suggest that there are at least three carboxylate ligands whose structures are significantly perturbed by Ca(2+)/Sr(2+) exchange. They are (1) the carboxylate ligand having a bridging or unidentate structure in the S(2) and S(3) states and perturbed in the S(1) --> S(2) and S(3) --> S(0) transitions, (2) that with a chelating or bridging structure in the S(1) and S(0) states and perturbed also in the S(1) --> S(2) and S(3) --> S(0) transitions, and (3) that with a chelating structure in the S(3) and S(0) states and changes in the S(2) --> S(3) and S(0) --> S(1) transitions. Taking into account the recent FTIR studies using site-directed mutagenesis and/or isotope substitution [Chu et al. (2004) Biochemistry 43, 3152-3116; Kimura et al. (2005) J. Biol. Chem. 280, 2078-2083; Strickler et al. (2006) Biochemistry 45, 8801-8811], it was concluded that these carboxylate groups do not originate from either D1-Ala344 (C-terminus) or D1-Glu189, which are located near the Ca(2+) ion in the X-ray crystallographic model of the Mn cluster. It was thus proposed that if the X-ray model is correct, the above carboxylate groups sensitive to Sr(2+) substitution are ligands to the Mn ions strongly coupled to the Ca(2+) ion rather than direct ligands to Ca(2+).     

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)(*).     

FTIR detection of structural changes in a histidine ligand during S-state cycling of photosynthetic oxygen-evolving complex

Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.     

Flash-induced Fourier transform infrared detection of the structural changes during the S-state cycle of the oxygen-evolving complex in photosystem II

Fourier transform infrared (FTIR) difference spectra of all flash-induced S-state transitions of the oxygen-evolving complex were measured using photosystem II (PSII) core complexes of Synechococcus elongatus. The PSII core sample was given eight successive flashes with 1 s intervals at 10 degrees C, and FTIR difference spectra upon individual flashes were measured. The obtained difference spectra upon the first to fourth flashes showed considerably different spectral features from each other, whereas the fifth, sixth, seventh, and eighth flash spectra were similar to the first, second, third, and fourth flash spectra, respectively. The intensities at the wavenumbers of prominent peaks of the first and second flash spectra showed clear period four oscillation patterns. These oscillation patterns were well fitted with the Kok model with 13% misses. These results indicate that the first, second, third, and fourth flash spectra represent the difference spectra upon the S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions, respectively. In these spectra, prominent bands were observed in the symmetric (1300-1450 cm(-)(1)) and asymmetric (1500-1600 cm(-)(1)) stretching regions of carboxylate groups and in the amide I region (1600-1700 cm(-)(1)). Comparison of the band features suggests that the drastic coordination changes of carboxylate groups and the protein conformational changes in the S(1) --> S(2) and S(2) --> S(3) transitions are reversed in the S(3) --> S(0) and S(0) --> S(1) transitions. The flash-induced FTIR measurements during the S-state cycle will be a promising method to investigate the detailed molecular mechanism of photosynthetic oxygen evolution.     

Light-induced FTIR difference spectroscopy as a powerful tool toward understanding the molecular mechanism of photosynthetic oxygen evolution

The molecular mechanism of photosynthetic oxygen evolution remains a mystery in photosynthesis research. Although recent X-ray crystallographic studies of the photosystem II core complex at 3.0-3.5 A resolutions have revealed the structure of the oxygen-evolving center (OEC), with approximate positions of the Mn and Ca ions and the amino acid ligands, elucidation of its detailed structure and the reactions during the S-state cycle awaits further spectroscopic investigations. Light-induced Fourier transform infrared (FTIR) difference spectroscopy was first applied to the OEC in 1992 as detection of its structural changes upon the S(1)-->S(2) transition, and spectra during the S-state cycle induced by consecutive flashes were reported in 2001. These FTIR spectra provide extensive structural information on the amino acid side groups, polypeptide chains, metal core, and water molecules, which constitute the OEC and are involved in its reaction. FTIR spectroscopy is thus becoming a powerful tool in investigating the reaction mechanism of photosynthetic oxygen evolution. In this mini-review, the measurement method of light-induced FTIR spectra of OEC is introduced and the results obtained thus far using this technique are summarized.     

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.     

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.     

Mid- to low-frequency Fourier transform infrared spectra of S-state cycle for photosynthetic water oxidation in Synechocystis sp. PCC 6803

Flash-induced Fourier transform infrared (FTIR) difference spectra for the four-step S-state cycle and the effects of global (15)N- and (13)C-isotope labeling on the difference spectra were examined for the first time in the mid- to low-frequency (1200-800 cm(-1)) as well as the mid-frequency (1700-1200 cm(-1)) regions using photosystem (PS) II core particles from cyanobacterium Synechocystis sp. PCC 6803. The difference spectra clearly exhibited the characteristic vibrational features for each transition during the S-state cycling. It is likely that the bands that change their sign and intensity with the S-state advances reflect the changes of the amino acid residues and protein matrices that have functional and/or structural roles within the oxygen-evolving complex (OEC). Except for some minor differences, the trends of S-state dependence in the 1700-1200 cm(-1) frequency spectra of the PS II cores from Synechocystis were comparable to that of spinach, indicating that the structural changes of the polypeptide backbones and amino acid side chains that occur during the oxygen evolution are inherently identical between cyanobacteria and higher plants. Upon (13)C-labeling, most of the bands, including amide I and II modes and carboxylate stretching modes, showed downward shifts; in contrast, (15)N-labeling induced isotopic shifts that were predominantly observed in the amide II region. In the mid- to low-frequency region, several bands in the 1200-1140 cm(-1) region were attributable to the nitrogen- and/or carbon-containing group(s) that are closely related to the oxygen evolution process. Specifically, the putative histidine ligand exhibited a band at 1113 cm(-1) which was affected by both (15)N- and (13)C-labeling and showed distinct S-state dependency. The light-induced bands in the 900-800 cm(-1) region were downshifted only by (13)C-labeling, whereas the bands in the 1000-900 cm(-1) region were affected by both (15)N- and (13)C-labeling. Several modes in the mid- to low-frequency spectra were induced by the change in protonation state of the buffer molecules accompanied by S-state transitions. Our studies on the light-induced spectrum showed that contributions from the redox changes of Q(A) and the non-heme iron at the acceptor side and Y(D) were minimal. It was, therefore, suggested that the observed bands in the 1000-800 cm(-1) region include the modes of the amino acid side chains that are coupled to the oxidation of the Mn cluster. S-state-dependent changes were observed in some of the bands.     

pH dependence of the flash-induced S-state transitions in the oxygen-evolving center of photosystem II from Thermosynechoccocus elongatus as revealed by Fourier transform infrared spectroscopy

pH dependence of the efficiencies of the flash-induced S-state transitions in the oxygen-evolving center (OEC) was studied by means of Fourier transform infrared (FTIR) difference spectroscopy using photosystem II (PSII) core complexes from the thermophilic cyanobacterium Thermosynechoccocus elongatus. The PSII core complexes dark-adapted at different pHs in the presence of ferricyanide as an electron acceptor were excited by four consecutive saturating laser flashes, and FTIR difference spectra induced by each flash were recorded in the region of 1800-1200 cm(-1). Each difference spectrum was fitted with a linear combination of standard spectra measured at pH 6.0, which represent the spectra upon individual S-state transitions, and the transition efficiencies were estimated from the fitting parameters. It was found that the S1 --> S2 transition probability is independent of pH throughout the pH region of 3.5-9.5, while the S2 --> S3, S3 --> S0, and S0 --> S1 transition probabilities decrease at acidic pH with pK values of 3.6 +/- 0.2, 4.2 +/- 0.3, and 4.7 +/- 0.5, respectively. These findings, i.e., the pH-independent S1 --> S2 transition probability and the pK values for the inhibition in the acidic range of the other three transitions, were in good agreement with recent results obtained by electron paramagnetic resonance measurements for PSII-enriched membranes of spinach [Bernát, G., Morvaridi, F., Feyziyev, Y., and Styring, S. (2002) Biochemistry 41, 5830-5843]. On the basis of this correspondence for quite different types of PSII preparations exhibiting marked difference in the pH dependence of the apparent proton release pattern, it is concluded that the inhibition of the S2 --> S3, S3 --> S0, and S0 --> S1 transitions in the acidic region is an inherent property of the OEC. This feature probably reflects proton release from substrate water in these three transitions. On the other hand, all of the S-state transitions remained generally efficient up to pH 9.5 in the alkaline region, except for a slight decrease of the S3 --> S0 transition probability above pH 8 (pK approximately 10). This observation partly differs from the tendency reported for spinach preparations, suggesting that a mechanism different from that in the acidic region is responsible for the transition efficiencies in the alkaline region.     

D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy

We report both mid-frequency (1800-1200 cm(-)(1)) and low-frequency (670-350 cm(-)(1)) S(2)/S(1) FTIR difference spectra of photosystem II (PSII) particles isolated from wild-type and D1-D170H mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Both mid- and low-frequency S(2)/S(1) spectra of the Synechocystis wild-type PSII particles closely resemble those from spinach PSII samples, which confirms an earlier result by Noguchi and co-workers [Noguchi, T., Inoue, Y., and Tang, X.-S. (1997) Biochemistry 36, 14705-14711] and indicates that the coordination environment of the oxygen evolving complex (OEC) in Synechocystis is very similar to that in spinach. We also found that there is no appreciable difference between the mid-frequency S(2)/S(1) spectra of wild-type and of D1-D170H mutant PSII particles, from which we conclude that D1-Asp170 does not undergo a significant structural change during the S(1) to S(2) transition. This result also suggests that, if D1-Asp170 ligates Mn, it does not ligate the Mn ion that is oxidized during the S(1) to S(2) state transition. Finally, we found that a mode at 606 cm(-)(1) in the low-frequency wild-type S(2)/S(1) spectrum shifts to 612 cm(-)(1) in the D1-D170H mutant spectrum. Because this 606 cm(-)(1) mode has been previously assigned to an Mn-O-Mn cluster mode of the OEC [Chu, H.-A., Sackett, H., and Babcock, G. T. (2000) Biochemistry 39, 14371-14376], we conclude that D1-Asp170 is structurally coupled to the Mn-O-Mn cluster structure that gives rise to this band. Our results suggest that D1-Asp170 either directly ligates Mn or Ca(2+) or participates in a hydrogen bond to the Mn(4)Ca(2+) cluster. Our results demonstrate that combining FTIR difference spectroscopy with site-directed mutagenesis has the potential to provide insights into structural changes in Mn and Ca(2+) coordination environments in the different S states of the OEC.     

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.     

Evaluation of photosynthetic activities in thylakoid membranes by means of Fourier transform infrared spectroscopy

Light-induced Fourier transformed infrared (FTIR) difference spectroscopy is a powerful method to study the structures and reactions of redox cofactors involved in the photosynthetic electron transport chain. So far, most of the FTIR studies of the reactions of oxygenic photosynthesis have been performed using isolated photosystem I (PSI) and photosystem II (PSII) preparations, which, however, could be modified during isolation procedures. In this study, we developed a methodology to evaluate the photosynthetic activities of thylakoids using FTIR spectroscopy. FTIR difference spectra upon successive flashes using thylakoids from spinach exhibited signals typical of the S-state cycle at the Mn₄CaO₅ cluster and Q_B reactions in PSII with period-four and -two oscillations, respectively. Similar measurement in the presence of an artificial quinone as an exogenous electron acceptor showed features specific to the S-state cycle. Simulations of the oscillation patterns provided the quantum efficiencies of the S-state cycle and electron transfer in PSII. Moreover, FTIR measurement under continuous illumination on thylakoids in the presence of DCMU showed signals due to Q_A reduction and P700 oxidation simultaneously. From the relative amplitudes of marker bands of Q_A^? and P700⁺, the molar ratio of photoactive PSII and PSI centers in thylakoids was estimated. FTIR analyses of the photo-reactions in thylakoids, which are more intact than isolated photosystems, will be useful in investigations of the photosynthetic mechanism especially by genetic modification of photosystem proteins.     

Perturbations at the chloride site during the photosynthetic oxygen-evolving cycle

Photosystem II (PSII) catalyzes the oxidation of water to O₂ at the manganese-containing, oxygen-evolving complex (OEC). Photoexcitation of PSII results in the oxidation of the OEC; four sequential oxidation reactions are required for the generation and release of molecular oxygen. Therefore, with flash illumination, the OEC cycles among five S _n states. Chloride depletion inhibits O₂ evolution. However, the binding site of chloride in the OEC is not known, and the role of chloride in oxygen evolution has not as yet been elucidated. We have employed reaction-induced FT-IR spectroscopy and selective flash excitation, which cycles PSII samples through the S state transitions. On the time scale employed, these FT-IR difference spectra reflect long-lived structural changes in the OEC. Bromide substitution supports oxygen evolution and was used to identify vibrational bands arising from structural changes at the chloride-binding site. Contributions to the vibrational spectrum from bromide-sensitive bands were observed on each flash. Sulfate treatment led to an elimination of oxygen evolution activity and of the FT-IR spectra assigned to the S₃ to S₀ (third flash) and S₀ to S₁ transitions (fourth flash). However, sulfate treatment changed, but did not eliminate, the FT-IR spectra obtained with the first and second flashes. Solvent isotope exchange in chloride-exchanged samples suggests flash-dependent structural changes, which alter protein dynamics during the S state cycle. Supported by NSF MCB 03-55421.     

Trapping of metalloradical intermediates of the S-states at liquid helium temperatures. Overview of the phenomenology and mechanistic implications

The oxygen-evolving complex (OEC) of photosystem II (PSII) consists of a Mn cluster (believed to be tetranuclear) and a tyrosine (Tyr Z or Y(Z)). During the sequential absorption of four photons by PSII, the OEC undergoes four oxidative transitions, S(0) to S(1), ..., S(3) to (S(4))S(0). Oxygen evolves during the S(3) to S(0) transition (S(4) being a transient state). Trapping of intermediates of the S-state transitions, particularly those involving the tyrosyl radical, has been a goal of ultimate importance, as that can test critically models employing a role of Tyr Z in proton (in addition to electron) transfer, and also provide important clues about the mechanism of water oxidation. Until very recently, however, critical experimental information was lacking. We review and evaluate recent observations on the trapping of metalloradical intermediates of the S-state transitions, at liquid helium temperatures. These transients are assigned to Tyr Z(*) magnetically interacting with the Mn cluster. Besides the importance of trapping intermediates of this unique catalytic mechanism, liquid helium temperatures offer the additional advantage that proton motions (unlike electron transfer) are blocked except perhaps across strong hydrogen bonds. This paper summarizes the recent observations and discusses the constraints that the phenomenology imposes.     

No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.     

Mutation of arginine 357 of the CP43 protein of photosystem II severely impairs the catalytic S-state cycle of the H2O oxidation complex

Basic amino acid side chains situated in active sites may mediate critical proton transfers during an enzymatic catalytic cycle. In the case of photosynthetic water oxidation, a strong base is postulated to facilitate the deprotonation of the active site Mn4-Ca cluster, thereby allowing the otherwise thermodynamically constrained transfer of an electron away from the Mn4-Ca cluster to the oxidized redox active tyrosine radical, YZ*, generated by photosynthetic charge separation. Arginine 357 of the CP43 polypeptide may be located in the second coordination shell of the O2-evolving Mn4-Ca cluster of photosystem II (PSII) according to current structural models. An ostensibly conservative substitution mutation, CP43-357K, was investigated using polarographic and fluorescence techniques in evaluating its potential impact on S-state cycling. Cells containing the CP43-357K mutation lost their capacity for autotrophic growth and exhibited a drastic reduction in O2 evolving activity ( approximately 15% of that of the wild type) despite the fact that mutant cells contained more than 80% of the concentration of charge-separating PSII reaction centers and more than half of these contained photooxidizable Mn. Fluorescence kinetics indicated that acceptor side electron transfer, dominated by the transfer of electrons from QA- to QB, was unaffected, but the fraction of centers containing Mn clusters capable of forming the S2 state was reduced to approximately 40% of that of the wild type. Analysis of O2 yields using a bare platinum electrode indicated a severe defect in the S-state cycling properties of the mutant H2O oxidation complexes. Although O2 evolution was delayed to the third flash during a train of single-turnover saturating flashes, the pattern of O2 emission did not exhibit a discernible periodicity indicating a very high miss factor, which was estimated to be approximately 45% compared to the wild-type value of approximately 10%. On the other hand, the multiflash fluorescence measurements indicate that the yield of formation of the S2 state from S1 is diminished by approximately 20%, although this latter estimate is complicated by the presence of damaged PSII centers. Taken together, the experiments indicate that the high miss factor observed during S-state cycling is likely due to a defect in the higher S-state transitions. These results are discussed in relation to the idea that CP43-R357 may serve as a ligand to bicarbonate or as the catalytic base proposed to mediate proton-coupled electron transfer (PCET) in the higher S states of the catalytic cycle of H2O oxidation.     

Flash-induced FTIR difference spectra of the water oxidizing complex in moderately hydrated photosystem II core films: effect of hydration extent on S-state transitions

Differently hydrated films of photosystem II (PSII) core complexes from Synechococcus elongatus were prepared in a humidity-controlled infrared cell. The relative humidity was changed by a simple method of placing a different ratio of glycerol/water solution in the sealed cell. The extent of hydration of the PSII film was lowered as the glycerol ratio increased. FTIR difference spectra of the water oxidizing complex upon the first to sixth flashes were measured at 10 degrees C using these hydrated PSII films. The FTIR spectra (1800-1200 cm(-1)) of the PSII films hydrated using 20% and 40% glycerol/water showed basically the same features as those of the core sample in solution [Noguchi, T., and Sugiura, M. (2001) Biochemistry 40, 1497-1502], and the prominent peaks exhibited clear period four oscillation patterns. These observations indicate that the S-state cycle properly functions in these hydrated samples. In the PSII films less hydrated, however, the efficiencies of S-state transitions decreased as the extent of hydration was lowered. This tendency was more significant in the S2 --> S3 and S3 --> S0 transitions than in the S1 --> S2 and S0 --> S1 transitions, indicating that the reactions or movements of water molecules are more strongly coupled with the former two transitions than the latter two. The implication of this observation was discussed in light of the water oxidizing mechanism especially in respect to the steps of substrate incorporation and proton release. Furthermore, in the OH stretching region (3800-3000 cm(-1)) of the first-flash spectrum, a differential signal was observed at 3618/3585 cm(-1), which was previously found in the S2/S1 spectrum of a frozen sample at 250 K and assigned to the water vibrations [Noguchi, T., and Sugiura, M. (2000) Biochemistry 39, 10943-10949]. The fact that the signal appeared even in rather dehydrated PSII films at a physiological temperature (10 degrees C) supported the idea that this water is located in the close vicinity of the Mn cluster and directly involved in the water oxidizing reaction. The results also showed that moderate hydration of the PSII sample made the whole OH region measurable, escaping from absorption saturation by bulk water, and thus will be a useful technique to monitor the water reactions during the S-state cycle using FTIR spectroscopy.     

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.     

The S0 state of photosystem II induced by hydroxylamine: differences between the structure of the manganese complex in the S0 and S1 states determined by X-ray absorption spectroscopy

Hydroxylamine at low concentrations causes a two-flash delay in the first maximum flash yield of oxygen evolved from spinach photosystem II (PSII) subchloroplast membranes that have been excited by a series of saturating flashes of light. Untreated PSII membrane preparations exhibit a multiline EPR signal assigned to a manganese cluster and associated with the S2 state when illuminated at 195 K, or at 273 K in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). We used the extent of suppression of the multiline EPR signal observed in samples illuminated at 195 K to determine the fraction of PSII reaction centers set back to a hydroxylamine-induced S0-like state, which we designate S0*. The manganese K-edge X-ray absorption edges for dark-adapted PSII preparations with or without hydroxylamine are virtually identical. This indicates that, despite its high binding affinity to the oxygen-evolving complex (OEC) in the dark, hydroxylamine does not reduce chemically the manganese cluster within the OEC in the dark. After a single turnover of PSII, a shift to lower energy is observed in the inflection of the Mn K-edge of the manganese cluster. We conclude that, in the presence of hydroxylamine, illumination causes a reduction of the OEC, resulting in a state resembling S0. This lower Mn K-edge energy of S0*, relative to the edge of S1, implies the storage and stabilization of an oxidative equivalent within the manganese cluster during the S0----S1 state transition. An analysis of the extended X-ray absorption fine structure (EXAFS) of the S0* state indicates that a significant structural rearrangement occurs between the S0* and S1 states. The X-ray absorption edge position and the structure of the manganese cluster in the S0* state are indicative of a heterogeneous mixture of formal valences of manganese including one Mn(II) which is not present in the S1 state.