Ammonia-induced structural changes of the oxygen-evolving complex in photosystem II as revealed by light-induced FTIR difference spectroscopy

Light-induced Fourier transform infrared difference spectroscopy has been applied to studies of ammonia effects on the oxygen-evolving complex (OEC) of photosystem II (PSII). We found that NH(3) induced characteristic spectral changes in the region of the symmetric carboxylate stretching modes (1450-1300 cm(-1)) of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra of PSII. The S(2) state carboxylate mode at 1365 cm(-1) in the S(2)Q(A)(-)/S(1)Q(A) spectrum of the controlled samples was very likely upshifted to 1379 cm(-1) in that of NH(3)-treated samples; however, the frequency of the corresponding S(1) carboxylate mode at 1402 cm(-1) in the same spectrum was not significantly affected. These two carboxylate modes have been assigned to a Mn-ligating carboxylate whose coordination mode changes from bridging or chelating to unidentate ligation during the S(1) to S(2) transition [Noguchi, T., Ono, T., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200; Kimura, Y., and Ono, T.-A. (2001) Biochemistry 40, 14061-14068]. Therefore, our results show that NH(3) induced significant structural changes of the OEC in the S(2) state. In addition, our results also indicated that the NH(3)-induced spectral changes of the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the temperature of the FTIR measurement. Among the temperatures we measured, the strongest effect was seen at 250 K, a lesser effect was seen at 225 K, and little or no effect was seen at 200 K. Furthermore, our results also showed that the NH(3) effects on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are dependent on the concentrations of NH(4)Cl. The NH(3)-induced upshift of the 1365 cm(-1) mode is apparent at 5 mM NH(4)Cl and is completely saturated at 100 mM NH(4)Cl concentration. Finally, we found that CH(3)NH(2) has a small but clear effect on the spectral change of the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum of PSII. The effects of amines on the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectra (NH(3) > CH(3)NH(2) > AEPD and Tris) are inverse proportional to their size (Tris approximately AEPD > CH(3)NH(2) > NH(3)). Therefore, our results showed that the effects of amines on the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII are sterically selective for small amines. On the basis of the correlations between the conditions (dependences on the excitation temperature and NH(3) concentration and the steric requirement for the amine effects) that give rise to the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum of PSII and the conditions that give rise to the altered S(2) state multiline EPR signal, we propose that the NH(3)-induced upshift of the 1365 cm(-1) mode is caused by the binding of NH(3) to the site on the Mn cluster that gives rise to the altered S(2) state multiline EPR signal. In addition, we found no significant NH(3)-induced change in the S(2)Q(A)(-)/S(1)Q(A) FTIR difference spectrum at 200 K. Under this condition, the OEC gives rise to the NH(3)-stabilized g = 4.1 EPR signal and a suppressed g = 2 multiline EPR signal. Our results suggest that the structural difference of the OEC between the normal g = 2 multiline form and the NH(3)-stabilized g = 4.1 form is small.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

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.     

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.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca2+ depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S(1)-to-S(2) transition was obtained with Ca(2+)-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca(2+) to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S(2)/S(1) FTIR spectrum upon Ca(2+) depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca(2+) depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K(+) at the Ca(2+) site. In the present study, the preparation of the Ca(2+)-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K(+). The obtained S(2)/S(1) spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm(-1) and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K(+). This result indicates that the drastic FTIR changes are a pure effect of Ca(2+) depletion, and provides solid evidence for the general view that Ca(2+) is strongly coupled with the Mn cluster.     

Flash-induced FTIR difference spectroscopy shows no evidence for the structural coupling of bicarbonate to the oxygen-evolving Mn cluster in photosystem II

Bicarbonate is known to be required for the maximum activity of photosystem II. Although it is well established that bicarbonate is bound to the nonheme iron to regulate the quinone reactions, the effect of bicarbonate on oxygen evolution is still controversial, and its binding site and exact physiological roles remain to be clarified. In this study, the structural coupling of bicarbonate to the oxygen-evolving center (OEC) was studied using Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra during the S-state cycle of OEC were recorded using the PSII core complexes from Thermosynechococcus elongatus in the presence of either unlabeled bicarbonate or (13)C-bicarbonate. The H (12)CO 3 (-)-minus-H (13)CO 3 (-) double difference spectra showed prominent bicarbonate bands at the first flash, whereas no appreciable bands were detected at the second to fourth flashes. The bicarbonate bands at the first flash were virtually identical to those from the nonheme iron, which was preoxidized by ferricyanide and photoreduced by a single flash, recorded using Mn-depleted PSII complexes. Using the bicarbonate bands of the nonheme iron as an internal standard, it was concluded that no bicarbonate band arising from OEC exists in the S-state FTIR spectra. This conclusion indicates that bicarbonate is not affected by the structural changes in OEC upon the four S-state transitions. It is thus strongly suggested that bicarbonate is neither a ligand to the Mn cluster nor a cofactor closely coupled to OEC, although the possibility cannot be fully excluded that nonexchangeable bicarbonate exists in OEC as a constituent of the Mn-cluster core. The data also provide strong evidence that bicarbonate does not function as a substrate or a catalytic intermediate. Bicarbonate may play major roles in the photoassembly process of the Mn cluster and in the stabilization of OEC by a rather indirect interaction.     

Structure of a histidine ligand in the photosynthetic oxygen-evolving complex as studied by light-induced fourier transform infrared difference spectroscopy.

Fourier transform infrared (FTIR) signals of a histidine side chain were identified in flash-induced S(2)/S(1) difference spectra of the oxygen-evolving complex (OEC) of photosystem II (PS II) using PS II membranes from globally (15)N-labeled spinach and PS II core complexes from Synechocystis cells in which both the imidazole nitrogens of histidine were selectively labeled with (15)N. A negative band at 1113-1114 cm(-1) was downshifted by 7 cm(-1) upon both global (15)N-labeling and selective [(15)N]His labeling, and assigned to the C-N stretching mode of the imidazole ring. This band was unaffected by H-D exchange in the PS II preparations. In addition, several peaks observed at 2500-2850 cm(-1) all downshifted upon global and selective (15)N-labeling. These were ascribed to Fermi resonance peaks on a hydrogen-bonding N-H stretching band of the histidine side chain. FTIR measurements of model compounds of the histidine side chain showed that the C-N stretching band around 1100 cm(-)(1) can be a useful IR marker of the protonation form of the imidazole ring. The band appeared with frequencies in the following order: Npi-protonated (>1100 cm(-1)) > imidazolate > imidazolium > Ntau-protonated (<1095 cm(-1)). The frequency shift upon N-deuteration was occurred in the following order: imidazolium (15-20 cm(-1)) > Ntau-protonated (5-10 cm(-1)) > Npi-protonated approximately imidazolate ( approximately 0 cm(-1)). On the basis of these findings together with the Fermi resonance peaks at >2500 cm(-1) as a marker of N-H hydrogen-bonding, we concluded that the histidine residue in the S(2)/S(1) spectrum is protonated at the Npi site and that this Npi-H is hydrogen bonded. This histidine side chain probably ligated the redox-active Mn ion at the Ntau site, and thus, oxidation of the Mn cluster upon S(2) formation perturbed the histidine vibrations, causing this histidine to appear in the S(2)/S(1) difference spectrum.     

Detection of structural changes upon S1-to-S2 transition in the oxygen-evolving manganese cluster in photosystem II by light-induced Fourier transform infrared difference spectroscopy.

The light-induced Fourier transform infrared (FT-IR) difference spectrum between the S1 and S2 states of the O2-evolving photosystem II (PSII) was obtained for the first time. Detection of an S2/S1 difference spectrum virtually free from contributions by the acceptor-side signals was achieved by employing an exogenous electron acceptor, potassium ferricyanide, to trypsin-treated PSII membranes and using one-flash excitation at 250 K. A synthetic difference spectrum obtained by adding this S2/S1 spectrum to the QA-/QA spectrum measured with Mn-depleted PSII was almost identical with the difference spectrum of the S2QA-/S1QA charge separation measured with untreated PSII. This successful simulation verifies the correctness of the S2/S1 spectrum thus obtained. The observed S2/S1 spectrum reflects the structural changes within the water-oxidizing Mn cluster upon the S1-to-S2 transition, most probably changes in vibrational modes of ligands coordinating to the Mn ion(s) that is (are) oxidized upon the S2 formation and/or changes in protein conformation. The present results demonstrate that FT-IR difference spectroscopy is a promising method to investigate the structure of the intermediates of the Mn cluster involved in photosynthetic water oxidation.     

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.     

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.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography

PsbY is one of the low molecular mass subunits of oxygen-evolving photosystem II (PSII). Its location, however, has not been identified in the current crystal structure of PSII. We constructed a PsbY-deletion mutant of Thermosynechococcus elongatus, crystallized, and analyzed the crystal structure of the mutant PSII dimer. The results obtained showed that PsbY is located in the periphery of PSII close to the alpha- and beta-subunits of cytochrome b559, which corresponded to an unassigned helix in the 3.7A structure of T. vulcanus or helix X2 in the 3.0A structure of T. elongatus. Our results also indicated that the C-terminal loop of PsbY is protruded toward the stromal side, instead of the lumenal side predicted previously.     

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.     

Light-induced FTIR difference spectroscopy of the S(2)-to-S(3) state transition of the oxygen-evolving complex in Photosystem II

We have applied flash-induced FTIR spectroscopy to study structural changes upon the S(2)-to-S(3) state transition of the oxygen-evolving complex (OEC) in Photosystem II (PSII). We found that several modes in the difference IR spectrum are associated with bond rearrangements induced by the second laser flash. Most of these IR modes are absent in spectra of S(2)/S(1), of the acceptor-side non-heme ion, of Yradical(D)/Y(D) and of S(3)'/S(2)' from Ca-depleted PSII preparations. Our results suggest that these IR modes most likely originate from structural changes in the oxygen-evolving complex itself upon the S(2)-to-S(3) state transition in PSII.     

Effects of ethylene glycol and methanol on ammonia-induced structural changes of the oxygen-evolving complex in photosystem II

Ammonia is an inhibitor of water oxidation and a structural analogue for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH(3)-specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH(3)-specific binding site on the OEC is estimated to be more than 10 times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH(3)-induced upshift of the 1365 cm(-)(1) IR mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH(3)-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations ( approximately 1 M), we conclude that ethylene glycol has a clear effect on the NH(3)-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum. In PSII samples containing 30% (v/v) ethylene glycol, only NH(3), not other bulkier amines (e.g., Tris, AEPD, and CH(3)NH(2)), has a clear effect on the upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH(3) and CH(3)NH(2) have a clear effect. On the basis of the results mentioned above, we propose that ethylene glycol acts directly or indirectly to decrease the affinity or limit the accessibility of NH(3) and CH(3)NH(2) to the NH(3)-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% (v/v) methanol does not have any significant effect on the NH(3)-induced upshift of the 1365 cm(-)(1) mode in the S(2)Q(A)(-)/S(1)Q(A) spectrum and the NH(3)-modified S(2) state g = 2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH(3) upon binding to the Mn site of the OEC that gives rise to the altered S(2) state g = 2 multiline EPR signal.     

Modified molecular interactions of the pheophytin and plastoquinone electron acceptors in photosystem II of chlorophyll d-containing Acaryochloris marina as revealed by FTIR spectroscopy

Photosynthesis Research - Acaryochloris marina is a unique cyanobacterium that contains chlorophyll (Chl) d as a major pigment. Because Chl d has smaller excitation energy than Chl a used in...     

Digalactosyldiacylglycerol is required for stabilization of the oxygen-evolving complex in photosystem II

The galactolipid digalactosyldiacylglycerol (DGDG) is present in the thylakoid membranes of oxygenic photosynthetic organisms such as higher plants and cyanobacteria. Recent x-ray crystallographic analysis of protein-cofactor supercomplexes in thylakoid membranes revealed that DGDG molecules are present in the photosystem II (PSII) complex (four molecules per monomer), suggesting that DGDG molecules play important roles in folding and assembly of subunits in the PSII complex. However, the specific role of DGDG in PSII has not been fully clarified. In this study, we identified the dgdA gene (slr1508, a ycf82 homolog) of Synechocystis sp. PCC6803 that presumably encodes a DGDG synthase involved in the biosynthesis of DGDG by comparison of genomic sequence data. Disruption of the dgdA gene resulted in a mutant defective in DGDG synthesis. Despite the lack of DGDG, the mutant cells grew as rapidly as the wild-type cells, indicating that DGDG is not essential for growth in Synechocystis. However, we found that oxygen-evolving activity of PSII was significantly decreased in the mutant. Analyses of the PSII complex purified from the mutant cells indicated that the extrinsic proteins PsbU, PsbV, and PsbO, which stabilize the oxygen-evolving complex, were substantially dissociated from the PSII complex. In addition, we found that heat susceptibility but not dark-induced inactivation of oxygen-evolving activity was notably increased in the mutant cells in comparison to the wild-type cells, suggesting that the PsbU subunit is dissociated from the PSII complex even in vivo. These results demonstrate that DGDG plays important roles in PSII through the binding of extrinsic proteins required for stabilization of the oxygen-evolving complex.     

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-.     

Altered structure of the Mn4Ca cluster in the oxygen-evolving complex of photosystem II by a histidine ligand mutation

The effect of replacing a histidine ligand on the properties of the oxygen-evolving complex (OEC) and the structure of the Mn₄Ca cluster in Photosystem II (PSII) is studied by x-ray absorption spectroscopy using PSII core complexes from the Synechocystis sp. PCC 6803 D1 polypeptide mutant H332E. In the x-ray crystallographic structures of PSII, D1-His³³² has been assigned as a direct ligand of a manganese ion, and the mutation of this histidine ligand to glutamate has been reported to prevent the advancement of the OEC beyond the S₂Yz^• intermediate state. The manganese K-edge (1s core electron to 4p) absorption spectrum of D1-H332E shifts to a lower energy compared with that of the native WT samples, suggesting that the electronic structure of the manganese cluster is affected by the presence of the additional negative charge on the OEC of the mutant. The extended x-ray absorption spectrum shows that the geometric structure of the cluster is altered substantially from that of the native WT state, resulting in an elongation of manganese-ligand and manganese-manganese interactions in the mutant. The strontium-H332E mutant, in which calcium is substituted by strontium, confirms that strontium (calcium) is a part of the altered cluster. The structural perturbations caused by the D1-H332E mutation are much larger than those produced by any biochemical treatment or mutation examined previously with x-ray absorption spectroscopy. The substantial structural changes provide an explanation not only for the altered properties of the D1-H332E mutant but also the importance of the histidine ligand for proper assembly of the Mn₄Ca cluster.