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.     

Low-frequency fourier transform infrared spectroscopy of the oxygen-evolving complex in Photosystem II*

In this communication, we report our progress on the development of low-frequency Fourier transform infrared (FTIR) spectroscopic techniques to study metal-substrate and metal-ligand vibrational modes in the Photosystem II/oxygen-evolving complex (PS II/OEC). This information will provide important structural and mechanistic insight into the OEC. Strong water absorption in the low-frequency region (below 1000 cm⁻¹), a lack of suitable materials, and temperature control problems have limited previous FTIR spectroscopic studies of the OEC to higher frequencies (>1000 cm⁻¹). We have overcome these technical difficulties that have blocked access to the low-frequency region and have developed successive instruments that allow us to move deeper into the low-frequency region (down to 350 cm⁻¹), while increasing both data accumulation efficiency and S/N ratio. We have detected several low-frequency modes in the S₂/S₁spectrum that are specifically associated with these two states. Our results demonstrate the utility of FTIR techniques in accessing low-frequency modes in Photosystem II and in proteins generally. This revised version was published online in June 2006 with corrections to the Cover Date.     

Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 micros. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA- QB/QA QB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA- X+/QA X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA- QB/QA QB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs.     

Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.     

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.     

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.     

The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B)

In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the hundreds of microseconds range) and fast phase (microseconds to tens of microseconds range) in AM260(W-->C) RCs. We conclude that the unusually strong hydrogen bond between the carbonyl of QA and His M219 in the Rb. sphaeroides RC is not obligatory for efficient electron transfer from QA- to QB.     

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

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

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.     

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.     

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.     

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.     

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.     

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680+ and the quinone acceptors QA- to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, QA and QB, and the S2 state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of QA- reoxidation.     

Energetics of primary and secondary electron transfer in Photosystem II membrane particles of spinach revisited on basis of recombination-fluorescence measurements

Photon absorption by one of the roughly 200 chlorophylls of the plant Photosystem II (PSII) results in formation of an equilibrated excited state (Chl200*) and is followed by chlorophyll oxidation (formation of P680+) coupled to reduction of a specific pheophytin (Phe), then electron transfer from Phe- to a firmly bound quinone (QA), and subsequently reduction of P680+ by a redox-active tyrosine residue denoted as Z. The involved free-energy differences (DeltaG) and redox potentials are of prime interest. Oxygen-evolving PSII membrane particles of spinach were studied at 5 degrees C. By analyzing the delayed and prompt Chl fluorescence, we determined the equilibrium constant and thus free-energy difference between Chl200* and the [Z+,QA-] radical pair to be -0.43+/-0.025 eV, at 10 mus after the photon absorption event for PSII in its S(3)-state. On basis of this value and previously published results, the free-energy difference between P680* and [P680+,QA-] is calculated to be -0.50+/-0.04 eV; the free-energy loss associated with electron transfer from Phe to QA is found to be 0.34+/-0.04 eV. The given uncertainty ranges do not represent a standard deviation or likely error, but an estimate of the maximal error. Assuming a QA-/QA redox potential of -0.08 V, the following redox-potential estimates are obtained: +1.25 V for P680/P680+; +1.21 V for Z/Z+ (at 10 mus); -0.42 V for Phe-/Phe; -0.58 V for P680*/P680+.     

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.     

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.     

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.     

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.