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This study compares the properties of the extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex with characteristic features of proteins that are known to attain a ‘natively unfolded’ or a ‘molten globule’ structure. The analysis leads to the conclusion that the MSP in solution is most likely a ‘molten globule’ with well defined compact regions of β structure. The possible role of these structural peculiarities of MSP in solution for its function as important constituent of the WOC is discussed.

This study compares the properties of the extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex with characteristic features of proteins that are known to attain a ‘natively unfolded’ or a ‘molten globule’ structure. The analysis leads to the conclusion that the MSP in solution is most likely a ‘molten globule’ with well defined compact regions of β structure. The possible role of these structural peculiarities of MSP in solution for its function as important constituent of the WOC is discussed.

Intracellular carbonic anhydrases (CA) in aquatic photosynthetic organisms are involved in the CO2‐concentrating mechanism (CCM), which helps to overcome CO2limitation in the environment. In the green alga Chlamydomonas reinhardtii, this CCM is initiated and maintained by the pH gradient created across the chloroplast thylakoid membranes by photosystem (PS) II‐mediated electron transport. We show here that photosynthesis is stimulated by a novel, intracellular α‐CA bound to the chloroplast thylakoids. It is associated with PSII on the lumenal side of the thylakoid membranes. We demonstrate that PSII in association with this lumenal CA operates to provide an ample flux of CO2for carboxylation.

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) a bolishes the TL band appearing around −55°C (TL −55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around −30°C (TL −30). NBD treatment also abolishes the EPR signal II fastof oxidized tyrosine donor, Y 21and inhibits the electron transport from diphenylcarbazide to 2,6-dichlorophenol-indophenol. It is concluded that the TL −55and TL −30bands can be assigned to oxidized tyrosine (Y 2+) and histidine (His +) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680 +.

The rate of photoinhibition of photosystem II (PSII) activities (photoinduced change of chlorophyll fluorescence yield, ΔF, and photoreduction of 2,6-dichlorophenol-indophenol) in O 2-evolving pea subchloroplast membrane fragments in medium depleted of CO 2was considerably decreased upon addition of 5 mM NaHCO 3before the light treatment. A similar effect was observed when 100 μM MnCl 2was added before the photoinhibition. In PSII membrane fragments depleted of Mn, the pre-illumination led to irreversible loss of the capability of PSII to be reactivated by Mn 2+, and the rate of the photoinhibition was decreased by a factor of 2 or 5 if the pre-illumination was done in the presence of 0.2 μM MnCl 2(≈4 Mn per PSII reaction center) added alone or in combination with 5 mM NaHCO 3, respectively. A similar protective effect of bicarbonate was also revealed in the dark, during thermoinactivation of O 2-evolving PSII at 40°C: the rate of thermoinactivation of ΔF was decreased by a factor of 3 if 5 mM NaHCO 3was added to the medium. The results are consistent with the idea that bicarbonate is an essential component of the Mn-containing water-oxidizing complex of PSII, which decreases its susceptibility to photoinhibition and thermoinactivation.

The light‐induced Fourier transform infrared difference (FT‐IR) spectrum originating from the donor side of O2‐evolving photosystem (PS) II was obtained in non‐depleted and CO2‐depleted PSII membrane preparations. The observed spectrum free of contributions from the acceptor side signals was achieved by employing 2 mM/18 mM ferri‐/ferrocyanide as a redox couple. This spectrum showed main positive bands at 1589 and 1365 cm−1and negative bands at 1560, 1541, 1522 and 1507 cm−1. CO‐depleted PSII preparations showed a quite different spectrum. The main positive and negative bands disappeared after depletion of bicarbonate. The addition of bicarbonate partially restored those bands again. Comparison between difference FT‐IR spectra of untreated and bicarbonate‐depleted PSII membranes indicated that the positive bands at 1589 and 1365 cm−1can be assigned to COO−stretching modes from bicarbonate. The higher frequency corresponds to uas(COO−) and the lower frequency to us(COO−). 13C‐Labeling FT‐IR measurements confirmed these findings and also suggested that the negative band at 1560 cm−1can be ascribed to uas(COO−). The data are discussed in the framework of the suggestion that bicarbonate can be a ligand to the Mn‐containing water‐oxidizing complex of PSII.

In the thermoluminescence (TL) glow curve of photosystem II, particles depleted of manganese, a tyrosine modifier, 7‐chloro‐4‐nitrobenz‐2‐oxa‐1,3‐diazole (NBD) a bolishes the TL band appearing around −55°C (TL−55). Addition of a histidine modifier, diethylpyrocarbonate results in the disappearance of the band peaking around −30°C (TL−30). NBD treatment also abolishes the EPR signal IIfastof oxidized tyrosine donor, Y21and inhibits the electron transport from diphenylcarbazide to 2,6‐dichlorophenol‐indophenol. It is concluded that the TL−55and TL−30bands can be assigned to oxidized tyrosine (Y2+) and histidine (His+) residues, respectively, which participate in electron transfer from manganese to the reaction center of chlorophyll, P680+.

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over-expressed in E. colicells. The existance of two in vivo expression products, a protein with Mrabout 33.5 kDa, corresponding to the full-length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy-terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.

The structure of the primary electron donor of photosystem II, P680, is still under debate. It is not decided if it is composed of a chlorophyll (Chl) monomer or dimer. In this study, Fourier transform infrared (FTIR) spectroscopy was used to analyze the changes in the vibration modes occurring upon photooxidation of P680 in a Mn-depleted PS II preparation. It is demonstrated that illumination of the above in the presence of artificial electron acceptors results in a light-minus-dark absorbance change typical of the formation ofP680+. The light-minus-dark difference FTIR spectrum obtained under similar conditions is characterized by two negative peaks located at 1694 and 1652 or 1626 cm−1that can be assigned to the 9-keto groups of the P680 Chl, the latter band being indicative of a strongly associated group. These vibrations are shifted to 1714 and 1676 cm−1, respectively, in the positive features of the difference spectrum attributed to P680+. The occurrence of two pairs of bands attributed to 9-keto groups is discussed in terms of P680 being formed of a Chl dimer.

Hydrogen (H 2) evolution rates were measured by the gas chromatographic technique upon illumination of different subchloroplast preparations of higher plants without exogenous hydrogenase under anaerobic conditions. Subchloroplast preparations enriched in photosystem II (PS II) in the presence of an electron donor TMPD ( N,N,N′,N′-tetramethyl- p-phenylenediamine) are shown to have higher H 2-evolution rates (up to 30 nmol/mg Chl per h) than preparations enriched in PS I under the same conditions. The data on the suppression of H 2evolution by well-known inhibitors of PS II (dinoseb, atrazine) prove that the H 2photoproduction is sensitized by PS II reaction centers.

Hydrogen (H2) evolution rates were measured by the gas chromatographic technique upon illumination of different subchloroplast preparations of higher plants without exogenous hydrogenase under anaerobic conditions. Subchloroplast preparations enriched in photosystem II (PS II) in the presence of an electron donor TMPD (N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) are shown to have higher H2‐evolution rates (up to 30 nmol/mg Chl per h) than preparations enriched in PS I under the same conditions. The data on the suppression of H2evolution by well‐known inhibitors of PS II (dinoseb, atrazine) prove that the H2photoproduction is sensitized by PS II reaction centers.

Oxygen-evolving photosystem II particles (DT 20) isolated from pea chloroplasts by digitonin-Triton X-100 fractionation were photoinhibited with 150 W·m −2white light, at 20°C under three conditions: aerobic, anaerobic and strongly reducing ( Ehpoised to approx. −250 mV with dithionite). Hill reaction rate (H 2O → BQ) and variable fluorescence ( Fv) declined in parallel in all three cases with shortening half times: 30, 10 and 2.5 min, respectively. Light-induced absorbance changes at 685 nm characteristic of reversible photoaccumulation of reduced pheophytin ( Eh≈ −250 mV) remained essentially unchanged. We conclude that the three types of photoinhibitory treatment do not impair the separation of charges between chlorophyll P-680 and pheophytin in the photosystem II reaction center.

The rate of photoinhibition of photosystem II (PSII) activities (photoinduced change of chlorophyll fluorescence yield, ΔF, and photoreduction of 2,6‐dichlorophenol‐indophenol) in O2‐evolving pea subchloroplast membrane fragments in medium depleted of CO2was considerably decreased upon addition of 5 mM NaHCO3before the light treatment. A similar effect was observed when 100 μM MnCl2was added before the photoinhibition. In PSII membrane fragments depleted of Mn, the pre‐illumination led to irreversible loss of the capability of PSII to be reactivated by Mn2+, and the rate of the photoinhibition was decreased by a factor of 2 or 5 if the pre‐illumination was done in the presence of 0.2 μM MnCl2(≈4 Mn per PSII reaction center) added alone or in combination with 5 mM NaHCO3, respectively. A similar protective effect of bicarbonate was also revealed in the dark, during thermoinactivation of O2‐evolving PSII at 40°C: the rate of thermoinactivation of ΔF was decreased by a factor of 3 if 5 mM NaHCO3was added to the medium. The results are consistent with the idea that bicarbonate is an essential component of the Mn‐containing water‐oxidizing complex of PSII, which decreases its susceptibility to photoinhibition and thermoinactivation.

The barley chloroplast psbA gene encoding D1 protein, one of the main photosystem II components, has been over‐expressed in E. colicells. The existance of two in vivo expression products, a protein with Mrabout 33.5 kDa, corresponding to the full‐length precursor of the 32 kDa D1 mature form, and a truncated 29 kDa polypeptide was revealed. A modified D1 protein containing six histidine residues at the carboxy‐terminus was also obtained. After isolation and renaturation, the ability of the recombinant D1 protein to bind atrazine and pigments from barley thylakoids was demonstrated.

The structure of the primary electron donor of photosystem II, P680, is still under debate. It is not decided if it is composed of a chlorophyll (Chl) monomer or dimer. In this study, Fourier transform infrared (FTIR) spectroscopy was used to analyze the changes in the vibration modes occurring upon photooxidation of P680 in a Mn‐depleted PS II preparation. It is demonstrated that illumination of the above in the presence of artificial electron acceptors results in a light‐minus‐dark absorbance change typical of the formation ofP680+. The light‐minus‐dark difference FTIR spectrum obtained under similar conditions is characterized by two negative peaks located at 1694 and 1652 or 1626 cm−1that can be assigned to the 9‐keto groups of the P680 Chl, the latter band being indicative of a strongly associated group. These vibrations are shifted to 1714 and 1676 cm−1, respectively, in the positive features of the difference spectrum attributed to P680+. The occurrence of two pairs of bands attributed to 9‐keto groups is discussed in terms of P680 being formed of a Chl dimer.

Suppression of electron flow (and its subsequent restoration with 3–10 mM NaHCO3) on the donor side of photosystem II is shown upon either a partial depletion of pea subchloroplast membranes in bicarbonate or the addition of 5–20 μM formate. At higher concentrations (5 mM) formate induces the known ‘bicarbonate effect’ on the acceptor side of photosystem II. In preparations depleted of manganese the restoration of electron flow with 0.1–0.2 μM MnCl2(2–4 Mn per photosystem II reaction center) occurs only in the presence of bicarbonate and it is accompanied by an increased functional binding of manganese. Restoration of electron flow with diphenylcarbazide or NH2OH does not require the addition of NaHCO3. It is suggested that bicarbonate participates in the formation of the Mn‐cluster capable of water oxidation or serves as a substrate for the water‐oxidizing center.

The light-induced Fourier transform infrared difference (FT-IR) spectrum originating from the donor side of O 2-evolving photosystem (PS) II was obtained in non-depleted and CO 2-depleted PSII membrane preparations. The observed spectrum free of contributions from the acceptor side signals was achieved by employing 2 mM/18 mM ferri-/ferrocyanide as a redox couple. This spectrum showed main positive bands at 1589 and 1365 cm −1and negative bands at 1560, 1541, 1522 and 1507 cm −1. CO-depleted PSII preparations showed a quite different spectrum. The main positive and negative bands disappeared after depletion of bicarbonate. The addition of bicarbonate partially restored those bands again. Comparison between difference FT-IR spectra of untreated and bicarbonate-depleted PSII membranes indicated that the positive bands at 1589 and 1365 cm −1can be assigned to COO −stretching modes from bicarbonate. The higher frequency corresponds to u as(COO −) and the lower frequency to u s(COO −). 13C-Labeling FT-IR measurements confirmed these findings and also suggested that the negative band at 1560 cm −1can be ascribed to u as(COO −). The data are discussed in the framework of the suggestion that bicarbonate can be a ligand to the Mn-containing water-oxidizing complex of PSII.

Oxygen‐evolving photosystem II particles (DT 20) isolated from pea chloroplasts by digitonin‐Triton X‐100 fractionation were photoinhibited with 150 W·m−2white light, at 20°C under three conditions: aerobic, anaerobic and strongly reducing (Ehpoised to approx. −250 mV with dithionite). Hill reaction rate (H2O → BQ) and variable fluorescence (Fv) declined in parallel in all three cases with shortening half times: 30, 10 and 2.5 min, respectively. Light‐induced absorbance changes at 685 nm characteristic of reversible photoaccumulation of reduced pheophytin (Eh≈ −250 mV) remained essentially unchanged. We conclude that the three types of photoinhibitory treatment do not impair the separation of charges between chlorophyll P‐680 and pheophytin in the photosystem II reaction center.

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light‐induced O2evolution in the Hill reaction with SiMo (I50∼20, 3 and 45 μM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light-induced O 2evolution in the Hill reaction with SiMo ( I50∼20, 3 and 45 μM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH 2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

Removal of bicarbonate from spinach photosystem II BBY particles by means of washing in a CO 2-free medium results in the loss of their capability to accumulate the S 2multiline EPR signal upon continuous illumination at 190 K. Addition of 1 mM NaHCO 3before illumination leads to a 50–60% restoration of the multiline signal. Similarly, in BBY particles depleted of Mn by treatment with 1 M Tris-HCl (pH 8.0) and 0.5 M MgCl 2, re-addition of MnCl 2in the presence of 1 mM NaHCO 3results in a partial restoration (∼30%) of the S 2multiline EPR signal of the Mn cluster, while in the absence of NaHCO 3no restoration is observed. The results provide further evidence that bicarbonate is essential for maintaining the Mn-containing oxygen-evolving complex of PS II in a functionally active form.

Removal of bicarbonate from spinach photosystem II BBY particles by means of washing in a CO2‐free medium results in the loss of their capability to accumulate the S2multiline EPR signal upon continuous illumination at 190 K. Addition of 1 mM NaHCO3before illumination leads to a 50–60% restoration of the multiline signal. Similarly, in BBY particles depleted of Mn by treatment with 1 M Tris‐HCl (pH 8.0) and 0.5 M MgCl2, re‐addition of MnCl2in the presence of 1 mM NaHCO3results in a partial restoration (∼30%) of the S2multiline EPR signal of the Mn cluster, while in the absence of NaHCO3no restoration is observed. The results provide further evidence that bicarbonate is essential for maintaining the Mn‐containing oxygen‐evolving complex of PS II in a functionally active form.