Specific isotopic labeling and photooxidation-linked structural changes in the manganese-stabilizing subunit of photosystem II

Photosystem II (PSII) oxidizes water to molecular oxygen; the catalytic site is a cluster of four manganese ions. The catalytic site undergoes four sequential light-driven oxidation steps to form oxygen; these sequentially oxidized states are referred to as the Sn states, where n refers to the number of oxidizing equivalents stored. The extrinsic manganese stabilizing protein (MSP) of PSII influences the efficiency and stability of the manganese cluster, as well as the rates of the S state transitions. To understand how MSP influences photosynthetic water oxidation, we have employed isotope editing and difference Fourier transform infrared spectroscopy. MSP was expressed in Escherichia coli under conditions in which MSP aspartic and glutamic acid residues label at yields of 65 and 41%, respectively. Asparagine and glutamine were also labeled by this approach. GC/MS analysis was consistent with minimal scrambling of label into other amino acid residues and with no significant scrambling into the peptide bond. Selectively labeled MSP was then reconstituted to PSII, which had been stripped of native MSP. Difference Fourier transform infrared spectroscopy was used to probe the S1QA to S2QA- transition at 200 K, as well as the S1QB to S2QB- transition at 277 K. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese during the S1 to S2 transition.     

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.     

Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution

Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.     

Alterations in carboxylate ligation at the active site of photosystem II.

Photosystem II (PSII) is the photosynthetic enzyme catalyzing the oxidation of water and reduction of plastoquinone (Q). This reaction occurs at a catalytic site containing four manganese atoms and cycling among five oxidation states, the Sn states, where n refers to the number of oxidizing equivalents stored. Biochemical and spectroscopic techniques have been used previously to conclude that aspartate 170 in the D1 subunit influences the structure and function of the PSII active site (Boerner, R. J., Nguyen, A. P., Barry, B. A., and Debus, R. J. (1992) Biochemistry 31, 6660-6672). Substitution of glutamate for aspartate 170 resulted in an assembled manganese cluster, which was capable of enzymatic turnover, but at lower steady-state oxygen evolution rates. Here, we obtained the difference (light-minus-dark) Fourier transform IR spectrum associated with the S2Q--minus-S1Q transition by illumination of oxygen-evolving wild-type and DE170D1 PSII preparations at 200 K. These spectra are known to be dominated by contributions from carboxylic acid and carboxylate residues that are close to or ligating the manganese cluster. Substitution of glutamate for aspartate 170 results in alterations in the S2Q--minus-S1Q spectrum; the alterations are consistent with a change in carboxylate coordination to manganese or calcium. In particular, the spectra are consistent with a shift from bridging/bidentate carboxylates in wild-type PSII to unidentate carboxylate ligation in DE170D1 PSII.     

Time-resolved infrared detection of the proton and protein dynamics during photosynthetic oxygen evolution

Photosynthetic oxygen evolution by plants and cyanobacteria is performed by water oxidation at the Mn(4)CaO(5) cluster in photosystem II. The reaction is known to proceed via a light-driven cycle of five intermediates called S(i) states (i = 0-4). However, the detailed reaction processes during the intermediate transitions remain unresolved. In this study, we have directly detected the proton and protein dynamics during the oxygen-evolving reactions using time-resolved infrared spectroscopy. The time courses of the absorption changes at 1400 and 2500 cm(-1), which represent the reactions and/or interaction changes of carboxylate groups and the changes in proton polarizability of strong hydrogen bonds, respectively, were monitored upon flash illumination. The results provided experimental evidence that during the S(3) → S(0) transition, drastic proton rearrangement, most likely reflecting the release of a proton from the catalytic site, takes place to form a transient state before the oxidation of the Mn(4)CaO(5) cluster that leads to O(2) formation. Early proton movement was also detected during the S(2) → S(3) transition. These observations reveal the common mechanism in which proton release facilitates the transfer of an electron from the Mn(4)CaO(5) cluster in the S(2) and S(3) states that already accumulate oxidizing equivalents. In addition, relatively slow rearrangement of carboxylate groups was detected in the S(0) → S(1) transition, which could contribute to the stabilization of the S(1) state. This study demonstrates that time-resolved infrared detection is a powerful method for elucidating the detailed molecular mechanism of photosynthetic oxygen evolution by pursuing the reactions of substrate and amino acid residues during the S-state transitions.     

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.     

Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.     

Calcium ligation in photosystem II under inhibiting conditions

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

Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.     

Towards understanding the chemistry of photosynthetic oxygen evolution: dynamic structural changes, redox states and substrate water binding of the Mn cluster in photosystem II

This mini-review summarizes my postdoctoral research in the labs of T. Wydrzynski/C.B. Osmond, J.H.A. Nugent/M.C.W. Evans and V.K. Yachandra/K. Sauer/M.P. Klein. The results are reported in the context of selected data from the literature. Special emphasis is given to the mode of substrate water binding, Mn oxidation states and the structures of the Mn cluster in the four (meta)stable redox states of the oxygen evolving complex. The paper concludes with a working model for the mechanism of photosynthetic water oxidation that combines mu-oxo bridge oxidation in the S(3) state (V.K. Yachandra, K. Sauer, M.P. Klein, Chem. Rev. 96 (1996) 2927-2950) with O-O bond formation between two terminal Mn-hydroxo ligands during the S(3)-->(S(4))-->S(0) transition.     

N-terminal truncations of manganese stabilizing protein identify two amino acid sequences required for binding of the eukaryotic protein to photosystem II and reveal the absence of one binding-related sequence in cyanobacteria

Manganese stabilizing protein (MSP) is an intrinsically disordered extrinsic subunit of photosystem II that regulates the stability and kinetic performance of the tetranuclear manganese cluster that oxidizes water to oxygen. An earlier study showed that deletion of the (1)E-(3)G domain of MSP caused no loss of activity reconstitution, whereas deletion of the (4)K-(10)E domain reduced binding of the protein from 2 to 1 mol of MSP/mol of photosystem II and lowered activity reconstitution to about 50% of the control value [Popelkova et al. (2002) Biochemistry 41, 2702-2711]. In this work we present evidence that deletion of 13 or 14 amino acid residues from the MSP N-terminus (mutants DeltaS13M and DeltaK14M) does not interfere either with functional binding of one copy of MSP to photosystem II or with reconstitution of oxygen evolution activity to 50% of the control level. Both of these mutants exhibit nonspecific binding to photosystem II at higher protein concentrations. Truncation of the MSP sequence by 18 amino acids (mutant DeltaE18M), however, causes a loss of protein binding and activity reconstitution. This result demonstrates that the N-terminal domain (15)T-(18)E is required for binding of at least one copy of MSP to photosystem II. Analyses of CD spectra reveal changes in the structure of DeltaE18M (loss of beta-sheet, gain of unordered structure). Use of the information gained from these experiments in analyses of N-terminal sequences of MSP from a number of species indicates that higher plants and algae possess two recognition domains that are required for MSP binding to PSII, whereas cyanobacteria lack the first N-terminal domain found in eukaryotes. This may explain the absence of a second copy of MSP in the crystal structure of PSII from Synechococcus elongatus [Zouni et al. (2001) Nature 409, 739-743].     

pH-induced conformational changes in the soluble manganese-stabilizing protein of photosystem II

In this paper, we analyzed the pH-induced changes in the conformational states of the manganese-stabilizing protein (MSP) of photosystem II. Distinct conformational states of MSP were identified using fluorescence spectra, far-UV circular dichroism, and pressure-induced unfolding at varying suspension pH values, and four different conformational states of MSP were clearly distinguished using the center of fluorescence spectra mass when suspension pH was altered from 2 to 12. MSP was completely unfolded at a suspension pH above 11 and partly unfolded below a pH of 3. Analysis of the center of fluorescence spectral mass showed that the MSP structure appears stably folded around pH 6 and 4. The conformational state of MSP at pH 4 seems more stable than that at pH 6. Studies of peak positions of tryptophan fluorescence and MSP-bound 1-anilinonaphthalene-8-sulfonic acid fluorescence spectra supported this observation. A decrease in the suspension pH to 2 resulted in significant alterations in the MSP structure possibly because of protonation of unprotonated residues at lower pH, suggesting the existence of a large number of unprotonated amino acid residues at neutral pH possibly useful for proton transport in oxygen evolution. The acidic pH-induced conformational changes of MSP were reversible upon increase of pH to neutral pH; however, N-bromosuccinimide modification of tryptophan (Trp241) blocks the recovery of pH-induced conformational changes in MSP, implying that Trp241 is a key residue for the unfolded protein to form a functional structure. Thus, pH-induced structural changes of stable MSP (pH 6-4) may be utilized to analyze its functionality as a cofactor for oxygen evolution.     

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

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

Asymmetry of 13C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using Escherichia coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

Chloride cofactor in the photosynthetic oxygen-evolving complex studied by fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy, using midfrequency S2/S1 FTIR difference spectra, has been applied to studies of chloride cofactor in the photosynthetic oxygen-evolving complex (OEC) to determine the effects of Cl(-) depletion and monovalent anion substitution. Cl(-) depletion resulted in the disappearance of a large part of the amide I and II vibrational modes, and induced characteristic modification in the features of the stretching modes of the carboxylate ligands of the Mn cluster. The normal spectral features were largely restored by replenishment of Cl(-) except for some changes in amide bands. The overall features of Br(-) -, I(-) -, or NO3(-) -substituted spectra were similar to those of the Cl(-) -reconstituted spectrum, consistent with their ability to support oxygen evolution. In contrast, the spectrum was significantly altered by the replacement of Cl(-) with F- or CH3COO(-), which resulted in marked suppression and distortion of both the carboxylate and amide bands. The activity of oxygen evolution restored by NO3(-) was as high as that by Cl(-) when measured under limited light conditions, indicating that the NO3(-) -substituted OEC is fully active in oxygen evolution, although with a slow turnover rate. The double-difference spectrum between the 14NO3(-) -substituted and 15NO3- -substituted S2/S1 difference spectrum showed isotopic bands for asymmetric NO stretching mode in the region of 1400-1300 cm(-1) due to NO3(-) bound to the Cl(-) site. This demonstrated structural coupling between the Cl(-) site and the Mn cluster. A proposed model for the isotopic bands suggested that Cl(-) as well as NO3(-) is not directly associated with the Mn cluster and exists in a more symmetric configuration and weaker binding state in the S2 state than in the S1 state. These results also suggest that Cl(-) is required for changes in the structure of the specific carboxylate ligand of the Mn cluster as well as the peptide backbone of protein matrixes upon the transition from S1 to S2.     

Sucrose and glycerol effects on photosystem II

Photosystem II catalyzes the oxidation of water and the reduction of plastoquinone. The active site cycles among five oxidation states, which are called the S(n) states. PSII purification procedures include the use of the cosolvents, sucrose and/or glycerol, to stabilize water splitting activity and for cryoprotection. In this study, the effects of sucrose and glycerol on PSII were investigated. Sucrose addition was observed to stimulate the steady-state rate of oxygen evolution in the range from 0 to 1.35 M. Glycerol addition was observed to stimulate oxygen evolution in the range from 0 to 30%. Both cosolvents were observed to be inhibitory at higher concentrations. Sucrose addition was shown to have no effect on the rate of Q(A)(-) oxidation or on the K(M) for exogenous acceptor. PSII was then treated to remove extrinsic proteins. In these samples, sucrose addition stimulated activity, but glycerol addition was inhibitory at concentrations higher than approximately 0.5 M. This inhibitory effect of glycerol at relatively low concentrations is attributed to glycerol binding to the active site, when extrinsic subunits are not present. Reaction induced FTIR spectra, associated with the S(1) to S(2) transition of the water-oxidizing complex, exhibited significant differences throughout the 1,800-1,200 cm(-1) region, when glycerol- and sucrose-containing samples were compared. These measurements suggest a cosolvent-induced shift in the pK(A) of an aspartic or glutamic acid side chain, as well as structural changes at the active site. These structural alterations are attributed to a change in preferential hydration of the oxygen-evolving complex.     

Primary Conformation Change in Bacteriorhodopsin on Photoexcitation

Ultrafast dynamics of bacteriorhodopsin (bR) has been extensively studied experimentally and theoretically. However, there are several contradictory results reported, indicating that its detailed dynamics and initial process have not yet been fully clarified. In this work, changes in the amplitude and phase of molecular vibration in the isomerization process of bR were real-time probed simultaneously at 128 different wavelengths through intensity modulation of the electronic transition. Systematic information on the transient change in continuous spectrum extending from 505 nm (2.45 eV) to 675 nm (1.84 eV) showed different dynamics in each spectral region reflecting the difference in the excited states and intermediates dominating the dynamics during the photoisomerization. Careful analysis of the transient spectral changes and spectrograms calculated from the vibrational real-time traces elucidated that the primary event just after photoexcitation is the deformation of the retinal configuration, which decays within 30 fs near the C=N bond in the protonated Schiff base. The intensity of C=N stretching mode starts to decrease before the initiation of the frequency modulation of the C=C stretching mode. The C=C stretching mode frequency was modulated by a coupled torsion around the C₁₃=C₁₄ bond, leading to the photoisomerization around the bond. This study clarified the dynamics of the C=N and C=C stretching modes working as key vibration modes in the photoisomerization of bR. Furthermore, we have elucidated the modulation and decay dynamics of the C=C stretching mode in the photoreaction starting from H (Franck-Condon excited state) followed by I (twisted excited), and J (first intermediate) states.     

Evidence that the C-terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: an isotope-edited FTIR study

Isotope-edited FTIR difference spectroscopy was employed to determine if the C-terminal alpha-COO(-) group of the D1 polypeptide ligates the (Mn)(4) cluster in photosystem II (PSII) and, if so, if it ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 were propagated photoautotrophically in the presence of L-[1-(13)C]alanine or unlabeled ((12)C) L-alanine. In wild-type cells, both the C-terminal alpha-COO(-) group of the D1 polypeptide at D1-Ala344 and all alanine-derived peptide carbonyl groups will be labeled. In D1-A344G and D1-A344S mutant cells, the C-terminal alpha-COO(-) group of the D1 polypeptide will not be labeled because this group is no longer provided by alanine. The resultant S(2)-minus-S(1) FTIR difference spectra of purified wild-type and mutant PSII particles showed that one symmetric carboxylate stretching mode that is altered during the S(1) --> S(2) transition is sensitive to L-[1-(13)C]alanine-labeling in wild-type PSII particles but not in D1-A344G and D1-A344S PSII particles. Because the only carboxylate group that can be labeled in the wild-type PSII particles but not in the mutant PSII particles is the C-terminal alpha-COO(-) group of the D1 polypeptide, we assign the L-[1-(13)C]alanine-sensitive symmetric carboxylate stretching mode to the alpha-COO(-) group of D1-Ala344. In unlabeled wild-type PSII particles, this mode appears at approximately 1356 cm(-1) in the S(1) state and at approximately 1339 or approximately 1320 cm(-1) in the S(2) state. These frequencies are consistent with unidentate ligation of the (Mn)(4) cluster by the alpha-COO(-) group of D1-Ala344 in both the S(1) and S(2) states. The apparent 17-36 cm(-1) downshift in frequency in response to the S(1) --> S(2) transition is consistent with the alpha-COO(-) group of D1-Ala344 ligating a Mn ion whose charge increases during the S(1) --> S(2) transition. Accordingly, we propose that the alpha-COO(-) group of D1-Ala344 ligates the Mn ion that undergoes an oxidation during the S(1) --> S(2) transition. Control experiments were conducted with Mn-depleted wild-type PSII particles. These experiments showed that tyrosine Y(D) may be structurally coupled to the carbonyl oxygen of an alanine-derived peptide carbonyl group.     

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.     

Participation of glutamate-354 of the CP43 polypeptide in the ligation of manganese and the binding of substrate water in photosystem II

In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn(4)Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn(4)Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H(2)(18)O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S(3) state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S(1) state Mn-EXAFS spectrum, a slightly altered S(2) state multiline EPR signal, a substantially altered S(2)-minus-S(1) FTIR difference spectrum, and an unusually long lifetime for the S(2) state (>10 h) in a substantial fraction of reaction centers. In contrast, the S(2) state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S(2)-minus-S(1) FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with (15)N and specific labeling with l-[1-(13)C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO(-) group of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm(-1) in both the S(1) and S(2) states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S(2) state and that most of these can achieve the S(3) state, but no evidence for advancement beyond the S(3) state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn(4)Ca cluster in the S(2) state, the FTIR and H(2)(18)O exchange data show that the mutation strongly influences other properties of the Mn(4)Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S(1) to S(2) transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S(3) state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn(4)Ca cluster in the S(1) state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S(1) to S(2) transition. The H(2)(18)O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S(3) state.