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

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

Photosynthetic water oxidation in Synechocystis sp. PCC6803: mutations D1-E189K, R and Q are without influence on electron transfer at the donor side of photosystem II.

The oxygen-evolving manganese cluster (OEC) of photosynthesis is oxidised by the photochemically generated primary oxidant (P(+*)(680)) of photosystem II via a tyrosine residue (Y(Z), Tyr161 on the D1 subunit of Synechocystis sp. PCC6803). The redox span between these components is rather small and probably tuned by protonic equilibria. The very efficient electron transfer from Y(Z) to P(+*)(680) in nanoseconds requires the intactness of a hydrogen bonded network involving Y(Z), D1-His190, and presumably D1-Glu189. We studied photosystem II core particles from photoautotrophic mutants where the residue D1-E189 was replaced by glutamine, arginine and lysine which were expected to electrostatically differ from the glutamate in the wild-type (WT). Surprisingly, the rates of electron transfer from Y(Z) to P(+*)(680) as well as from the OEC to Y(ox)(Z) were the same as in the WT. With the generally assumed proximity between D1-His190 (and thus D1-Glu189) and Y(Z), the lack of any influence on the electron transfer around Y(Z) straightforwardly implies a strongly hydrophobic environment forcing Glu (acid) and Lys, Arg (basic) at position D1-189 into electro-neutrality. As one alternative, D1-Glu189 could be located at such a large distance from the OEC, Y(Z) and P(+*)(680) that a charge on D1-189X does not influence the electron transfer. This seems less likely in the light of the drastic influence of its direct neighbour, D1-His190, on Y(Z) function. Another alternative is that D1-Glu189 is negatively charged, but is located in a cluster of acid/base groups that compensates for an alteration of charge at position 189, leaving the overall net charge unchanged in the Gln, Lys, and Arg mutants.     

Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

Recent models for water oxidation in photosystem II postulate that the tyrosine Y(Z) radical, Y(Z)(*), abstracts both an electron and a proton from the Mn cluster during one or more steps in the catalytic cycle. This coupling of proton- and electron-transfer events is postulated to provide the necessary driving force for oxidizing the Mn cluster in its higher oxidation states. The formation of Y(Z)(*) requires the deprotonation of Y(Z) by His190 of the D1 polypeptide. For Y(Z)(*) to abstract both an electron and a proton from the Mn cluster, the proton abstracted from Y(Z) must be transferred rapidly from D1-His190 to the lumenal surface via one or more proton-transfer pathways. The proton acceptor for D1-His190 has been proposed to be either Glu189 of the D1 polypeptide or a group positioned by this residue. To further define the role of D1-Glu189, 17 D1-Glu189 mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. Several of these mutants are of particular interest because they appear to assemble Mn clusters in 70-80% of reaction centers in vivo, but evolve no O(2). The EPR and electron-transfer properties of PSII particles isolated from the D1-E189Q, D1-E189L, D1-E189D, D1-E189N, D1-E189H, D1-E189G, and D1-E189S mutants were examined. Intact PSII particles isolated from mutants that evolved no O(2) also exhibited no S(1) or S(2) state multiline EPR signals and were unable to advance beyond an altered Y(Z)(*)S(2) state, as shown by the accumulation of narrow "split" EPR signals under multiple turnover conditions. In the D1-E189G and D1-E189S mutants, the quantum yield for oxidizing the S(1) state Mn cluster was very low, corresponding to a > or =1400-fold slowing of the rate of Mn oxidation by Y(Z)(*). In Mn-depleted D1-Glu189 mutant PSII particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in the mutants was accelerated, showing that the mutations alter the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-Glu189 participating in a network of hydrogen bonds that modulates the properties of both Y(Z) and the Mn cluster and are consistent with proposals that D1-Glu189 positions a group that accepts a proton from D1-His190.     

Histidine 190-D1 and glutamate 189-D1 provide structural stabilization in photosystem II

In photosynthesis, photosystem II (PSII) conducts the light-driven oxidation of water to oxygen. Tyrosine Z is Tyr 161 of the D1 polypeptide; Z acts as an intermediary electron carrier in water oxidation. In this report, EPR spectroscopy was used to study the effect of His 190 and Glu 189 on Z* yield and reduction kinetics. Neither mutation has a significant impact on the EPR line shape of Z*. At room temperature and pH 7.5, the E189Q-D1 mutation has a single turnover Z* yield that is 84% compared to wild-type. The H190Q-D1 mutation decreases the Z* yield at room temperature by a factor of 2.6 but has a more modest effect (factor of 1.6) at -10 degrees C. The temperature dependence is shown to be primarily reversible. Neither mutation has a dramatic effect on Z* decay kinetics. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on H190Q, reveals perturbations, including an increased spectral contribution from a PSII chlorophyll. The Z* minus Z FT-IR spectrum, recorded at pH 7.5 on E189Q, shows perturbations, including a decreased contribution from the carboxylate side chain of a glutamate or aspartate. Temperature-dependent changes in H190Q-D1 and E189Q-D1 Z. yield are attributed to a reversible conformational change, which alters the electron-transfer rate from Z to P(680)(+). On the basis of these results, we conclude that H190 and E189 play a role in the structural stabilization of PSII. We postulate that some or all of the phenotypic changes observed in H190Q and E189Q mutants may be caused by structural alterations in PSII.     

Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II

An electron spin-echo envelope modulation study [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708] and a recent Fourier transform infrared study [Noguchi, T., Inoue, Y., and Tang, X.-S. (1999) Biochemistry 38, 10187-10195], both conducted with [(15)N]histidine-labeled photosystem II particles, show that at least one histidine residue coordinates the O(2)-evolving Mn cluster in photosystem II. Evidence obtained from site-directed mutagenesis studies suggests that one of these residues may be His332 of the D1 polypeptide. The mutation D1-H332E is of particular interest because cells of the cyanobacterium Synechocystis sp. PCC 6803 that contain this mutation evolve no O(2) but appear to assemble Mn clusters in nearly all photosystem II reaction centers [Chu, H.-A., Nguyen, A. P. , and Debus, R. J. (1995) Biochemistry 34, 5859-5882]. Photosystem II particles isolated from the Synechocystis D1-H332E mutant are characterized in this study. Intact D1-H332E photosystem II particles exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the S(2) state multiline EPR signal observed in wild-type PSII particles. However, the quantum yield for oxidizing the S(1) state Mn cluster is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*), and the temperature threshold for forming the S(2) state is approximately 100 K higher than in wild-type PSII preparations. Furthermore, the D1-H332E PSII particles are unable to advance beyond the Y(Z)(*)S(2) state, as shown by the accumulation of a narrow "split" EPR signal under multiple turnover conditions. In Mn-depleted photosystem II particles, charge recombination between Q(A)(*)(-) and Y(Z)(*) in D1-H332E is accelerated in comparison to wild-type, showing that the mutation alters the redox properties of Y(Z) in addition to those of the Mn cluster. These results are consistent with D1-His332 being located near the Mn-Y(Z) complex and perhaps ligating Mn.     

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

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.     

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

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

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.     

Participation of the C-terminal region of the D1-polypeptide in the first steps in the assembly of the Mn4Ca cluster of photosystem II

Amino acid residue D1-Asp(170) of the D1-polypeptide of photosystem II was previously shown to be implicated in the binding and oxidation of the first manganese to be assembled into the Mn(4)Ca cluster of the oxygen-evolving complex (OEC). According to recent x-ray crystallographic structures of photosystem II, D1-Glu(333) is proposed to participate with D1-Asp(170) in the coordination of Mn4 of the OEC. Other residues in the C-terminal region of the D1-polypeptide are proposed to coordinate nearby manganese of the cluster. Site-directed replacements in Synechocystis sp. PCC 6803 at D1-His(332), D1-Glu(333), D1-Asp(342), D1-Ala(344), and D1-Ser(345) were examined with regard to their ability to influence the binding and oxidation of the first manganese in manganese-depleted photosystem II core complexes. Direct and indirect measurements reveal in all mutants, but most marked in D1-Glu(333) replaced by His, an impaired ability of Mn(2+) to reduce Y(Z)., indicating a reduced ability (elevated K(m)) compared with WT to bind and oxidize the first manganese of the OEC. The effect on the K(m) of these mutations is, however, considerably weaker than some of those constructed at D1-Asp(170) (replacement by Asn, Ala, and Ser). These observations imply that the C-terminal residues ultimately involved in manganese coordination contribute to the high affinity binding at D1-Asp(170) likely through electrostatic interactions. That these residues are far from D1-Asp(170) in the primary structure of the D1-polypeptide, imply that the C terminus of the D1-polypeptide is already close to its mature conformation at the first stages of assembly of the Mn(4)Ca cluster.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Influence of the redox potential of the primary quinone electron acceptor on photoinhibition in photosystem II

We report the characterization of the effects of the A249S mutation located within the binding pocket of the primary quinone electron acceptor, Q(A), in the D2 subunit of photosystem II in Thermosynechococcus elongatus. This mutation shifts the redox potential of Q(A) by approximately -60 mV. This mutant provides an opportunity to test the hypothesis, proposed earlier from herbicide-induced redox effects, that photoinhibition (light-induced damage of the photosynthetic apparatus) is modulated by the potential of Q(A). Thus the influence of the redox potential of Q(A) on photoinhibition was investigated in vivo and in vitro. Compared with the wild-type, the A249S mutant showed an accelerated photoinhibition and an increase in singlet oxygen production. Measurements of thermoluminescence and of the fluorescence yield decay kinetics indicated that the charge-separated state involving Q(A) was destabilized in the A249S mutant. These findings support the hypothesis that a decrease in the redox potential of Q(A) causes an increase in singlet oxygen-mediated photoinhibition by favoring the back-reaction route that involves formation of the reaction center chlorophyll triplet. The kinetics of charge recombination are interpreted in terms of a dynamic structural heterogeneity in photosystem II that results in high and low potential forms of Q(A). The effect of the A249S mutation seems to reflect a shift in the structural equilibrium favoring the low potential form.     

Electron spin-lattice relaxation of the S0 state of the oxygen-evolving complex in photosystem II and of dinuclear manganese model complexes

The temperature dependence of the electron spin-lattice relaxation time T1 was measured for the S0 state of the oxygen-evolving complex (OEC) in photosystem II and for two dinuclear manganese model complexes by pulse EPR using the inversion-recovery method. For [Mn(III)Mn(IV)(mu-O)2 bipy4]ClO4, the Raman relaxation process dominates at temperatures below 50 K. In contrast, Orbach type relaxation was found for [Mn(II)Mn(III)(mu-OH)(mu-piv)2(Me3 tacn)2](ClO4)2 between 4.3 and 9 K. For the latter complex, an energy separation of 24.7-28.0 cm(-1) between the ground and the first excited electronic state was determined. In the S0 state of photosystem II, the T1 relaxation times were measured in the range of 4.3-6.5 K. A comparison with the relaxation data (rate and pre-exponential factor) of the two model complexes and of the S2 state of photosystem II indicates that the Orbach relaxation process is dominant for the S0 state and that its first excited state lies 21.7 +/- 0.4 cm(-1) above its ground state. The results are discussed with respect to the structure of the OEC in photosystem II.     

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.     

Ligation of D1-His332 and D1-Asp170 to the manganese cluster of photosystem II from Synechocystis assessed by multifrequency pulse EPR spectroscopy

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S(2) state. Spectra of natural isotopic abundance PSII ((14)N-PSII), uniformly (15)N-labeled PSII ((15)N-PSII), and (15)N-PSII containing (14)N-histidine ((14)N-His/(15)N-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional K(a)- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new (14)N-based interactions.     

Electron paramagnetic resonance signals from the S(3) state of the oxygen-evolving complex. A broadened radical signal induced by low-temperature near-infrared light illumination

The tetranuclear manganese cluster responsible for the oxidation of water in photosystem II cycles through five redox states denoted S(i)() (i = 0, 1, 2, 3, 4). Progress has been made recently in the detection of weak low-field EPR absorptions in both the perpendicular and parallel modes, associated with the integer spin state S(3) [Matsukawa, T., Mino, H., Yoneda, D., and Kawamori, A. (1999) Biochemistry 38, 4072-4077]. We confirm observation of these signals and have obtained them in high yield by illumination of photosystem II membranes, in which the non-heme iron was chemically preoxidized. It is shown that a split g = 4 signal accompanies the S(3) state signals. The signals diminish in the presence of ethanol and vanish in the presence of methanol. This effect is similar to that exerted by these alcohols to the high-spin component (g = 4.1) of the S(2) state and suggests that the latter spin configuration is the precursor of the S(3) state low-field signals. The S(3) state shows similar sensitivity to infrared illumination as has been observed previously in the S(2) state [Boussac, A., Un, S., Horner, O., and Rutherford, A. W. (1998) Biochemistry 37, 4001-4007]. Illumination of the S(3) state with near-infrared light (700-900 nm), at temperatures around 50 K, results in the modification of the low-field signals and most notably to the appearance of a broad (DeltaH approximately 200 G) radical-type signal centered at g = 2. The signal is tentatively assigned to the interaction of the Mn cluster in a modified S(2) state with a radical.     

Chloride regulation of enzyme turnover: application to the role of chloride in photosystem II

Chloride-dependent α-amylases, angiotensin-converting enzyme (ACE), and photosystem II (PSII) are activated by bound chloride. Chloride-binding sites in these enzymes contain a positively charged Arg or Lys residue crucial for chloride binding. In α-amylases and ACE, removal of chloride from the binding site triggers formation of a salt bridge between the positively charged Arg or Lys residue involved in chloride binding and a nearby carboxylate residue. The mechanism for chloride activation in ACE and chloride-dependent α-amylases is 2-fold: (i) correctly positioning catalytic residues or other residues involved in stabilizing the enzyme-substrate complex and (ii) fine-tuning of the pKa of a catalytic residue. By using examples of how chloride activates α-amylases and ACE, we can gain insight into the potential mechanisms by which chloride functions in PSII. Recent structural evidence from cyanobacterial PSII indicates that there is at least one chloride-binding site in the vicinity of the oxygen-evolving complex (OEC). Here we propose that, in the absence of chloride, a salt bridge between D2:K317 and D1:D61 (and/or D1:E333) is formed. This can cause a conformational shift of D1:D61 and lower the pKa of this residue, making it an inefficient proton acceptor during the S-state cycle. Movement of the D1:E333 ligand and the adjacent D1:H332 ligand due to chloride removal could also explain the observed change in the magnetic properties of the manganese cluster in the OEC upon chloride depletion.     

Mechanism of photosynthetic water oxidation: combining biophysical studies of photosystem II with inorganic model chemistry

A mechanism for photosynthetic water oxidation is proposed based on a structural model of the oxygen-evolving complex (OEC) and its placement into the modeled structure of the D1/D2 core of photosystem II. The structural model of the OEC satisfies many of the geometrical constraints imposed by spectroscopic and biophysical results. The model includes the tetranuclear manganese cluster, calcium, chloride, tyrosine Z, H190, D170, H332 and H337 of the D1 polypeptide and is patterned after the reversible O2-binding diferric site in oxyhemerythrin. The mechanism for water oxidation readily follows from the structural model. Concerted proton-coupled electron transfer in the S2-->S3 and S3-->S4 transitions forms a terminal Mn(V)=O moiety. Nucleophilic attack on this electron-deficient Mn(V)=O by a calcium-bound water molecule results in a Mn(III)-OOH species, similar to the ferric hydroperoxide in oxyhemerythrin. Dioxygen is released in a manner analogous to that in oxyhemerythrin, concomitant with reduction of manganese and protonation of a mu-oxo bridge.     

Nitric oxide-induced formation of the S-2 state in the oxygen-evolving complex of photosystem II from Synechococcus elongatus

In spinach photosystem II (PSII) membranes, the tetranuclear manganese cluster of the oxygen-evolving complex (OEC) can be reduced by incubation with nitric oxide at -30 degrees C to a state which is characterized by an Mn(2)(II, III) EPR multiline signal [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581-3587]. This state was recently assigned to the S(-)(2) state of the OEC [Schansker, G., Goussias, C., Petrouleas, V., and Rutherford, A. W. (2002) Biochemistry 41, 3057-3064]. On the basis of EPR spectroscopy and flash-induced oxygen evolution patterns, we show that a similar reduction process takes place in PSII samples of the thermophilic cyanobacterium Synechococcus elongatus at both -30 and 0 degrees C. An EPR multiline signal, very similar but not identical to that of the S(-)(2) state in spinach, was obtained with monomeric and dimeric PSII core complexes from S. elongatus only after incubation at -30 degrees C. The assignment of this EPR multiline signal to the S(-)(2) state is corroborated by measurements of flash-induced oxygen evolution patterns and detailed fits using extended Kok models. The small reproducible shifts of several low-field peak positions of the S(-)(2) EPR multiline signal in S. elongatus compared to spinach suggest that slight differences in the coordination geometry and/or the ligands of the manganese cluster exist between thermophilic cyanobacteria and higher plants.     

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.