Far-Red Light Accelerates Photosynthesis in the Low-Light Phases of Fluctuating Light.

It is well known that far-red light (FR; >700 nm) drives PSI photochemistry, but its effect on photosynthetic performance has received little attention. In this study, the effects of the addition of FR to red fluctuating light (FL) have on photosynthesis were examined in the leaves of Arabidopsis thaliana. Light-activated leaves were illuminated with FL [alternating high light/low light (HL/LL) at 800/30 µmol m-2 s-1] for 10-15 min without or with FR at intensities that reflected natural conditions. The CO2 assimilation rates upon the transition from HL to LL were significantly greater with FR than without FR. The enhancement of photosynthesis by FR was small under the steady-state conditions and in the HL phases of FL. Proton conductivity through the thylakoid membrane (gH+) in the LL phases of FL, estimated from the dark relaxation kinetics of the electrochromic absorbance shift, was greater with FR than without FR. The relaxation of non-photochemical quenching (NPQ) in the PSII antenna system and the increase in PSII photochemistry in the LL phases accelerated in the presence of FR. Similar FR-effects in FL were confirmed in typical sun and shade plants. On the basis of these results, we concluded that FR exerted beneficial effects on photosynthesis in FL by exciting PSI and accelerating NPQ relaxation and PSII-yield increase. This was probably because of the increased gH+, which would reflect faster ΔpH dissipation and ATP synthesis.

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy.

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the ß subunits. However, these changes are not observed in the isolated ß chain (ß4 homotetramer) and, therefore, are associated with the α-ß inter-subunit interactions. For the communications between α and ß subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or ß Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.

Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260-nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260-nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or ß subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(ßH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260-nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by (1) H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe-His F8 linkage in both α and ß subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual (1) H NMR technique. Chirality 28:585-592, 2016. © 2016 Wiley Periodicals, Inc.

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and ß Subunits in the α2ß2 Tetramer.

Human hemoglobin (Hb), which is an α2ß2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by 'cooperativity'. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and ß subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and ß subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or ß subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(ßH92G), in which only the Fe-His in the ß subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(ßH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the ß subunit simply enhances the O2-affinity of α subunit.

Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII.

Involvement of propionate side chains of the heme in circular dichroism of myoglobin: experimental and theoretical analyses.

Incorporation of the heme into globin induces a prominent circular dichroism (CD) band in the Soret region. The appearance of heme optical activity is widely believed to arise from the interaction between the heme and aromatic residues of the globin. However, hemoglobin (Hb) containing the reversed heme exhibits a CD spectrum obviously different from that of native Hb, indicating that the interactions of heme side chains with globin contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native myoglobin (Mb) and those of Mb reconstituted with synthetic hemes lacking vinyl and/or propionate. Replacement of 2,4-vinyl groups with methyl induced moderate changes. In contrast, replacement of 6,7-propionate groups with carboxylate resulted in complete disappearance of the positive Soret CD band. To get theoretical basis for the contributions of 6,7-side chains on the band, we investigated the CD spectra at a time-dependent density functional theory level. In the antiparallel conformation of the 6,7-side chains, the rotational strengths were calculated to be positive, on the other hand in the parallel conformation to be negative. We also found that the weak Soret CD band in 2,4-dimethyl-6,7-dicarboxyheme can be explained by canceling between different carboxyl conformers.

Cyanobacterial monogalactosyldiacylglycerol-synthesis pathway is involved in normal unsaturation of galactolipids and low-temperature adaptation of Synechocystis sp. PCC 6803.

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of "normal growth conditions" when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity.

Psb28 is involved in recovery of photosystem II at high temperature in Synechocystis sp. PCC 6803.

Psb28 is an extrinsic protein of photosystem II (PSII), which is conserved among photosynthetic organisms from cyanobacteria to higher plants. A unicellular cyanobacterium, Synechocystis sp. PCC 6803, has two homologs of Psb28, Psb28-1 and Psb28-2. However, the role of these proteins remains poorly understood. In this study, we disrupted the psb28-1 (sll1398) and psb28-2 (slr1739) genes in wild-type Synechocystis sp. PCC 6803 and examined their photosynthetic properties to elucidate the physiological role of Psb28 in photosynthesis. We also disrupted the psb28-1 gene in a dgdA mutant defective in the biosynthesis of digalactosyldiacylglycerol, in which Psb28-1 significantly accumulates in PSII. The disruption of the psb28-1 gene in the wild-type resulted in growth retardation under high-light conditions at high temperatures with a low rate of restoration of photodamaged photosynthetic machinery. Similar phenomena were observed at normal growth temperatures in the psb28-1/dgdA double mutant. In contrast, disruption of psb28-2 in the wild-type and dgdA mutant did not affect host strain phenotype, suggesting that Psb28-2 does not contribute to the recovery of PSII. In addition, protein analysis using strains expressing His-tagged Psb28-1 revealed that Psb28-1 is mainly associated with the CP43-less PSII monomer. In the dgdA mutant, the CP43-less PSII monomer accumulated to a greater extent than in the wild-type, and its accumulation caused greater accumulation of Psb28-1 in PSII. These results demonstrate that Psb28-1 plays an important role in PSII repair through association with the CP43-less monomer, particularly at high temperatures.

The role of lipids in photosystem II.

The thylakoid membranes of photosynthetic organisms, which are the sites of oxygenic photosynthesis, are composed of monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG). The identification of many genes involved in the biosynthesis of each lipid class over the past decade has allowed the generation and isolation of mutants of various photosynthetic organisms incapable of synthesizing specific lipids. Numerous studies using such mutants have revealed that deficiency of these lipids primarily affects the structure and function of photosystem II (PSII) but not of photosystem I (PSI). Recent X-ray crystallographic analyses of PSII and PSI complexes from Thermosynechococcus elongatus revealed the presence of 25 and 4 lipid molecules per PSII and PSI monomer, respectively, indicating the enrichment of lipids in PSII. Therefore, lipid molecules bound to PSII may play special roles in the assembly and functional regulation of the PSII complex. This review summarizes our present understanding of the biochemical and physiological roles of lipids in photosynthesis, with a special focus on PSII. This article is part of a Special Issue entitled: Photosystem II.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination.

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1) s(-1)) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature.

The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 degrees C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 degrees C to 38 degrees C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits.

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni2+-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b(6)f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in Synechocystis sp. PCC 6803.

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38 degrees C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38 degrees C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.

Lack of digalactosyldiacylglycerol increases the sensitivity of Synechocystis sp. PCC 6803 to high light stress.

The physiological role of digalactosyldiacylglycerol (DGDG) in photosynthesis was examined using a dgdA mutant of Synechocystis sp. PCC 6803 that is defective in the biosynthesis of DGDG. The dgdA mutant cells showed normal growth under low light (LL) conditions. However, their growth was retarded under high light (HL) conditions and under Ca(2+)- and/or Cl(-)-limited conditions compared to wild-type cells. The retardation in growth of the mutant cells was recovered by exogenous supply of DGDG in the growth medium. The dgdA mutant showed increased sensitivity to photoinhibition. Although both photodamage and repair processes of photosynthesis were affected, the repair process was more severely affected than the photodamage process, suggesting that DGDG plays an important role in the photosynthetic repair cycle.

A method for analyzing lipid-modified proteins with mass spectrometry.

In protein analysis using mass spectrometry, proteins are usually separated by electrophoresis and digested within the gel with proteases such as trypsin. However, analysis of lipid-modified proteins is difficult due to the low recovery of lipid-modified peptide fragments from the gel as well as their low ionization efficiency during mass spectrometry. In this study, we developed a simple extraction method with n-dodecyl-beta-D-maltoside following chloroform/methanol extraction that efficiently elutes lipid-modified fragments from gels. This method allowed us to analyze the structure of lipid-modified fragments, suggesting the applicability of the method for analysis of lipid-modified fragments by mass spectrometry.

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

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

Effects of the lack of phosphatidylglycerol on the donor side of photosystem II.

Our previous studies with the pgsA mutant of the cyanobacterium Synechocystis sp. PCC6803 (hereafter termed pgsA mutant), which is defective for the biosynthesis of phosphatidylglycerol (PG), revealed an important role for PG in the electron acceptor side of photosystem II (PSII), especially in the electron transport between plastoquinones Q(A) and Q(B). This study now shows that PG also plays an important role in the electron donor side of PSII, namely, the oxygen-evolving system. Analyses of purified PSII complexes indicated that PSII from PG-depleted pgsA mutant cells sustained only approximately 50% of the oxygen-evolving activity compared to wild-type cells. Dissociation of the extrinsic proteins PsbO, PsbV, and PsbU, which are required for stabilization of the manganese (Mn) cluster, followed by the release of a Mn atom, was observed in PSII of the PG-depleted mutant cells. The released PsbO rebound to PSII when PG was added back to the PG-depleted mutant cells, even when de novo protein synthesis was inhibited. Changes in photosynthetic activity of the PG-depleted pgsA mutant cells induced by heat treatment or dark incubation resembled those of DeltapsbO, DeltapsbV, and DeltapsbU mutant cells. These results suggest that PG plays an important role in binding extrinsic proteins required for sustaining a functional Mn cluster on the donor side of PSII.

FTIR detection of structural changes in a histidine ligand during S-state cycling of photosynthetic oxygen-evolving complex.

Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.

Changes in structural and functional properties of oxygen-evolving complex induced by replacement of D1-glutamate 189 with glutamine in photosystem II: ligation of glutamate 189 carboxylate to the manganese cluster.

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at approximately 70% of the rate of control wild-type particles. The E189Q OEC showed typical S(2) state electron spin resonance signals, and the spin center distance between the S(2) state manganese cluster and the Y(D) (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S(2) state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S(1) to S(2) transition.