FTIR detection of water reactions during the flash-induced S-state cycle of the photosynthetic water-oxidizing complex

Photosynthetic water oxidation is performed via the light-driven S-state cycle in the water-oxidizing complex (WOC) of photosystem II (PS II). To understand its molecular mechanism, monitoring the reaction of substrate water in each S-state transition is essential. We have for the first time detected the reactions of water molecules in WOC throughout the S-state cycle by observing the OH vibrations of water using flash-induced Fourier transform infrared (FTIR) difference spectroscopy. Moderately hydrated (or deuterated) PS II core films from Synechococcus elongatus were used to obtain the FTIR difference spectra upon the first, second, third, and fourth flash illumination, representing the structural changes in the S(1) --> S(2), S(2) --> S(3), S(3) --> S(0), and S(0) --> S(1) transitions, respectively. In the weakly H-bonded OH region, bands appeared at 3617/3588 cm(-1) as a differential signal in the first-flash spectrum and at 3634, 3621, and 3612 cm(-1) with negative intensities in the second-, third-, and fourth-flash spectra, respectively. These bands shifted down by approximately 940 cm(-1) upon deuteration and by approximately 10 cm(-1) upon H(18)O substitution, indicating that they arise from the OH stretches of water including the substrate and its intermediates. Strongly D-bonded OD bands of water were also identified as broad features in the range of 2600-2200 cm(-1) by taking the double difference between the spectra of D(2)(16)O- and D(2)(18)O-deuterated films. In addition, broad continuum features that probably arise from the large proton polarizability of H-bonds were observed around 3000, 2700, 2550, and 2600 cm(-1) in the first-, second-, third-, and fourth-flash spectra, respectively, of the hydrated PS II film, revealing changes in the H-bond network of the protein. The negative OH intensities upon the second to fourth flashes might be related to proton release from substrate water. The results presented here showed that FTIR detection of water OH(D) bands can be a powerful method for investigating the mechanism of photosynthetic water 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.     

FTIR detection of water reactions in the oxygen-evolving centre of photosystem II

Flash-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the water-oxidizing reactions in the oxygen-evolving centre of photosystem II. Reactions of water molecules were directly monitored by detecting the OH stretching bands of weakly H-bonded OH of water in the 3700-3500 cm(-1) region in FTIR difference spectra during S-state cycling. In the S1-->S2 transition, a band shift from 3588 to 3617 cm(-1) was observed, indicative of a weakened H-bond. Decoupling experiments using D2O:H2O (1:1) showed that this OH arose from a water molecule with an asymmetric H-bonding structure and this asymmetry became more significant upon S2 formation. In the S2-->S3, S3-->S0 and S0-->S1 transitions, negative bands were observed at 3634, 3621 and 3612 cm(-1), respectively, representing formation of a strong H-bond or a proton release reaction. In addition, using complex spectral features in the carboxylate stretching region (1600-1300 cm-(1)) as 'fingerprints' of individual S-state transitions, pH dependency of the transition efficiencies and the effect of dehydration were examined to obtain the information of proton release and water insertion steps in the S-state cycle. Low-pH inhibition of the S2-->S3, S3-->S0 and S0-->S1 transitions was consistent with a view that protons are released in the three transitions other than S1-->S2, while relatively high susceptibility to dehydration in the S2-->S3 and S3-->S0 transitions suggested the insertion of substrate water into the system during these transitions. Thus, a possible mechanism of water oxidation to explain the FTIR data is proposed.     

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.     

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.     

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.     

No evidence from FTIR difference spectroscopy that aspartate-170 of the D1 polypeptide ligates a manganese ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.     

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.     

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+).     

Analysis of flash-induced FTIR difference spectra of the S-state cycle in the photosynthetic water-oxidizing complex by uniform 15N and 13C isotope labeling

Protein bands in flash-induced Fourier transform infrared (FTIR) difference spectra of the S-state cycle of photosynthetic water oxidation were analyzed by uniform (15)N and (13)C isotopic labeling of photosystem II (PS II). The difference spectra upon first- to fourth-flash illumination were obtained with hydrated (for the 1800-1200 cm(-)(1) region) or deuterated (for the 3500-3100 cm(-)(1) region) films of unlabeled, (15)N-labeled, and (13)C-labeled PS II core complexes from Thermosynechococcus elongatus. Shifts of band frequencies upon (15)N and (13)C labeling provided the assignments of major peaks in the regions of 3450-3250 and 1700-1630 cm(-)(1) to the NH stretches and amide I modes of polypeptide backbones, respectively, and the assignments of some of the peaks in the 1600-1500 cm(-)(1) region to the amide II modes of backbones. Other prominent peaks in the latter region and most of the peaks in the 1450-1300 cm(-)(1) region exhibited large downshifts upon (13)C labeling but were unchanged by (15)N labeling, and hence assigned to the asymmetric and symmetric COO(-) stretching vibrations, respectively, of carboxylate groups in Glu, Asp, or the C-terminus. Peak positions corresponded well with each other among the first- to fourth-flash spectra, and most of the bands in the first- and/or second-flash spectra appeared with opposite signs of intensity in the third- and/or fourth-flash spectra. This observation indicates that the protein movements in the S(1)-->S(2) and/or S(2)-->S(3) transitions are mostly reversed in the S(3)-->S(0) and/or S(0)-->S(1) transitions, representing a catalytic role of the protein moieties of the water-oxidizing complex. Drastic structural changes in carboxylate groups over the S-state cycle suggest that the Asp and/or Glu side chains play important roles in the reaction mechanism of photosynthetic water oxidation.     

Water-sensitive low-frequency vibrations of reaction intermediates during S-state cycling in photosynthetic water oxidation

In photosynthetic water oxidation, two water molecules are converted to an oxygen molecule through five reaction intermediates, designated S(n) (n = 0-4), at the catalytic Mn cluster of photosystem II. To understand the mechanism of water oxidation, changes in the chemical nature of the substrate water as well as the Mn cluster need to be defined during S-state cycling. Here, we report for the first time a complete set of Fourier transform infrared difference spectra during S-state cycling in the low-frequency (670-350 cm(-1)) region, in which interactions between the Mn cluster and its ligands can be detected directly, in PS II core particles from Thermosynechococcus elongatus. Furthermore, vibrations from oxygen and/or hydrogen derived from the substrate water and changes in them during S-state cycling were identified using multiplex isotope-labeled water, including H2(18)O, D2(16)O, and D2(18)O. Each water isotope affected the low-frequency S-state cycling spectra, characteristically. The bands sensitive only to (16)O/(18)O exchange were assigned to the modes from structures involving Mn and oxygen having no interactions with hydrogen, while the bands sensitive only to H/D exchange were assigned to modes from amino acid side chains and/or polypeptide backbones that associate with water hydrogen. The bands sensitive to both (16)O/(18)O and H/D exchanges were attributed to the structure involving Mn and oxygen structurally coupled with hydrogen in a direct or an indirect manner through hydrogen bonds. These bands include the changes of intermediate species derived from substrate water during the process of photosynthetic water oxidation.     

No evidence from FTIR difference spectroscopy that aspartate-342 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, Asp342 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn4 cluster. To determine if D1-Asp342 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions, the FTIR difference spectra of the individual S state transitions in D1-D342N mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that the mid-frequency (1800-1200 cm-1) FTIR difference spectra of wild-type and D1-D342N PSII particles are essentially identical. Importantly, the mutation alters none of the carboxylate vibrational modes that are present in the wild-type spectra. The absence of significant mutation-induced spectral alterations in D1-D342N PSII particles shows that the oxidation of the Mn4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp342 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. One explanation of these data is that D1-Asp342 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-Asp342 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn4 cluster during the S1 --> S2 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 Mn4 cluster during the S0 --> S1 and S2 --> S3 transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Asp342, or D1-Ala344. In separate experiments that were conducted with l-[1-13C]alanine, we found no evidence that D1-Asp342 ligates the same Mn ion that is ligated by the alpha-COO- group of D1-Ala344.     

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.     

Structural and oxidation state changes of the photosystem II manganese complex in four transitions of the water oxidation cycle (S0 --> S1, S1 --> S2, S2 --> S3, and S3,4 --> S0) characterized by X-ray absorption spectroscopy at 20 K and room temperature

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.     

Structure of an active water molecule in the water-oxidizing complex of photosystem II as studied by FTIR spectroscopy

The vibrations of a water molecule in the water-oxidizing complex (WOC) of photosystem II were detected for the first time using Fourier transform infrared (FTIR) spectroscopy. In a flash-induced FTIR difference spectrum upon the S(1)-to-S(2) transition, a pair of positive and negative bands was observed at 3618 and 3585 cm(-1), respectively, and both bands exhibited downshifts by 12 cm(-1) upon replacement of H(2)(16)O by H(2)(18)O. Upon D(2)O substitution, the bands largely shifted down to 2681 and 2652 cm(-1). These observations indicate that the bands at 3618 and 3585 cm(-1) arise from the O-H stretching vibrations of a water molecule, probably substrate water, coupled to the Mn cluster in the S(2) and S(1) states, respectively. The band frequencies indicate that the O-H group forms a weak H-bond and this H-bonding becomes weaker upon S(2) formation. Intramolecular coupling with the other O-H vibration of this water molecule was studied by a decoupling experiment using a H(2)O/D(2)O (1:1) mixture. The downshifts by decoupling were estimated to be 4 and 12 cm(-1) for the 3618 (S(2)) and 3585 cm(-1) (S(1)) bands, both of which were much smaller than 52 cm(-1) of water in vapor, indicating that the observed water has a considerably asymmetric structure; i.e., one of the O-H groups is weakly and the other is strongly H-bonded. The smaller coupling in the S(2) than the S(1) state means that this H-bonding asymmetry becomes more prominent upon S(2) formation. Such a structural change may facilitate the proton release reaction that takes place in the later step by lowering the potential barrier. The present study showed that FTIR detection of the O-H vibrations is a useful and promising method to directly monitor the chemical reactions of substrate water and clarify the molecular mechanism of photosynthetic water oxidation.     

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.     

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.     

The multiplicity of roles for (bi)carbonate in photosystem II operation in the hypercarbonate-requiring cyanobacterium <Emphasis Type="Italic">Arthrospira maxima</Emphasis>

Arthrospira maxima is unique among cyanobacteria, growing at alkaline pH (<11) in concentrated (bi)carbonate (1.2 M saturated) and lacking carbonic anhydrases. We investigated dissolved inorganic carbon (DIC) roles within PSII of A. maxima cells oximetrically and fluorometrically, monitoring the light reactions on the donor and acceptor sides of PSII. We developed new methods for removing DIC based on a (bi)carbonate chelator and magnesium for (bi)carbonate ionpairing. We established relative affinities of three sites: the water-oxidizing complex (WOC), non-heme iron/Q_A^–, and solvent-accessible arginines throughout PSII. Full reversibility is achieved but (bi)carbonate uptake requires light. DIC depletion at the non-heme iron site and solvent-accessible arginines greatly reduces the yield of O₂ due to O₂ uptake, but accelerates the PSII–WOC cycle, specifically the S2→S3 and S3→S0 transitions. DIC removal from the WOC site abolishes water oxidation and appears to influence free energy stabilization of the WOC from a site between CP43-R357 and Ca²⁺.     

Efficiency and role of loss processes in light-driven water oxidation by PSII

Its superior quantum efficiency renders PSII a model for biomimetic systems. However, also in biological water oxidation by PSII, the efficiency is restricted by recombination losses. By laser-flash illumination, the secondary radical pair, P680(+)Q(-) (A) (where P680 is the primary Chl donor in PSII and Q(A), primary quinone acceptor of PSII), was formed in close to 100% of the PSII. Investigation of the quantum efficiency (or yield) of the subsequent steps by time-resolved delayed (10 micros to 60 ms) and prompt (70 micros to 700 ms) Chl fluorescence measurements on PSII membrane particles suggests that (1) the effective rate for P680(+) Q(-) (A) recombination is approximately 5 ms(-1) with an activation energy of approximately 0.34 eV, circumstantially confirming dominating losses by reformation of the primary radical pair followed by ground-state recombination. (2) Because of compensatory influences on recombination and forward reactions, the efficiency is only weakly temperature dependent. (3) Recombination losses are several-fold enhanced at lower pH. (4) Calculation based on delayed-fluorescence data suggests that the losses depend on the state of the water-oxidizing manganese complex, being low in the S(0)-->S(1) and S(1)-->S(2) transition, clearly higher in S(2)-->S(3) and S(3)-->S(4)-->S(0). (5) For the used artificial electron acceptor, the efficiency is limited by acceptor-side processes/S-state decay at high/low photon-absorption rates resulting in optimal efficiency at surprisingly low rates of approximately 0.15-15 photons s(-1) (per PSII). The pH and S-state dependence can be rationalized by the basic model of alternate electron-proton removal proposed elsewhere. A physiological function of the recombination losses could be limitation of the lifetime of the reactive donor-side tyrosine radical (Y(.) (Z)) in the case of low-pH blockage of water oxidation.     

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.