Biochimica et Biophysica Acta (BBA) - Bioenergetics
BBA reportBicarbonate, not CO2, is the species required for the stimulation of Photosystem II electron transport
Abstract
Evidence is presented that the bicarbonate ion (HCO−3), not CO2, H2CO3 or CO2−3, is the species that stimulates electron transport in Photosystem II from spinach (Spinacia oleracea). Advantage was taken of the pH dependence of the ratio of HCO−3 to CO2 at equilibrium in order to vary effectively the concentration of one species while holding the other constant. The Hill reaction was stimulated in direct proportion with the equilibrium HCO−3 concentration, but it was independent of the equilibrium CO2 concentration. The other two carbonic species, H2CO3 and CO2−3, are also shown to have no direct involvement. It is suggested that HCO−3 is the species which binds to the effector site.
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Cited by (33)
Photosystem II and the unique role of bicarbonate: A historical perspective
2012, Biochimica et Biophysica Acta - BioenergeticsIn photosynthesis, cyanobacteria, algae and plants fix carbon dioxide (CO2) into carbohydrates; this is necessary to support life on Earth. Over 50 years ago, Otto Heinrich Warburg discovered a unique stimulatory role of CO2 in the Hill reaction (i.e., O2 evolution accompanied by reduction of an artificial electron acceptor), which, obviously, does not include any carbon fixation pathway; Warburg used this discovery to support his idea that O2 in photosynthesis originates in CO2. During the 1960s, a large number of researchers attempted to decipher this unique phenomenon, with limited success. In the 1970s, Alan Stemler, in Govindjee's lab, perfected methods to get highly reproducible results, and observed, among other things, that the turnover of Photosystem II (PSII) was stimulated by bicarbonate ions (hydrogen carbonate): the effect would be on the donor or the acceptor, or both sides of PSII. In 1975, Thomas Wydrzynski, also in Govindjee's lab, discovered that there was a definite bicarbonate effect on the electron acceptor (the plastoquinone) side of PSII. The most recent 1.9 Å crystal structure of PSII, unequivocally shows HCO3− bound to the non-heme iron that sits in-between the bound primary quinone electron acceptor, QA, and the secondary quinone electron acceptor QB. In this review, we focus on the historical development of our understanding of this unique bicarbonate effect on the electron acceptor side of PSII, and its mechanism as obtained by biochemical, biophysical and molecular biological approaches in many laboratories around the World. We suggest an atomic level model in which HCO3−/CO32 − plays a key role in the protonation of the reduced QB. In addition, we make comments on the role of bicarbonate on the donor side of PSII, as has been extensively studied in the labs of Alan Stemler (USA) and Vyacheslav Klimov (Russia). We end this review by discussing the uniqueness of bicarbonate's role in oxygenic photosynthesis and its role in the evolutionary development of O2-evolving PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Interaction of bicarbonate with the manganese-stabilizing protein of photosystem II
2010, Journal of Photochemistry and Photobiology B: BiologyThe effect of reversible removal of on structural re-arrangements in the Mn-stabilizing protein (MSP) of photosystem II, isolated from pea leaves, was studied using measurements of characteristic alterations in fluorescence of hydrophobic probe 8-anilino-1-naphthalene-sulfonic acid (ANS). It was shown that the treatments capable of removal of (or CO2) from possible binding sites in MSP (pH lowering from 6.5 to 3.5, addition of a structurally similar anion in concentration 1–20 mM or air evacuation at pH 3.5) result in a significant (up to 370%) increase of ANS fluorescence (indicative of structural changes in MSP), whereas lowers the ANS fluorescence to the initial level observed in untreated protein at pH 6.5. Since the effects are revealed at (sub)micromolar concentrations of , the specific high-affinity binding of (or CO2) to MSP (required for its native structure preservation) is proposed. Possible bicarbonate binding sites and its physiological role within the water-oxidizing complex of photosystem II are discussed.
Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii
1998, Biochimica et Biophysica Acta - BioenergeticsFormate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA− persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA−QB↔QAQB−, in the direction of [QA−], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA− to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (∼70% of the wild type) level. (6) The Fo level was elevated (∼40–50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA−] in the dark. (7) A decreased (∼10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants.
Modification of the photosystem II acceptor side function in a D1 mutant (arginine-269-glycine) of Chlamydomonas reinhardti
1997, Biochimica et Biophysica Acta - BioenergeticsBicarbonate anions have a strong positive influence on the electron and proton transfers in photosystem II (PS II). It has been suggested that bicarbonate binds to the non-heme iron and the QB binding niche of the PS II reaction center. To investigate the potential amino acid binding environment of bicarbonate, an arginine residue (R269) of the D1 protein of PS II of Chlamydomonas reinhardtii was mutated into a glycine; our characterization of the resultant mutant (D1-R269G) shows that both the Tyr+D and Q−A Fe2+ EPR signals are substantially reduced and assembly of the tetranuclear Mn is lost (R.S. Hutchison, J. Xiong, R.T. Sayre, Govindjee, Biochim. Biophys. Acta 1277 (1996) 83–92). In order to understand the molecular implications of this mutation on the electron acceptor side of PS II, we used chlorophyll (Chl) a fluorescence as a probe of PS II structure and function, and herbicide binding as a probe for changes in the QB binding niche of PS II. Chl fluorescence measurements with the heterotrophically grown D1-R269G mutant cells (or thylakoids), as compared to that of the wild type, show that: rate of electron transfer from Q−A to the plastoquinone pool, measured by flash-induced Chl a fluorescence decay kinetics, is reduced by ∼17 fold; the minimum Chl a fluorescence yield when all Q−A is oxidized, is elevated by 2 fold; the level of stable charge separation as inferred from variable Chl fluorescence is reduced by 44%; binary oscillation pattern of variable Chl a fluorescence obtained after a series of light flashes is absent, indicative of the loss of functioning of the two-electron gate on the PS II acceptor side; 77 K PS II Chl a fluorescence emission bands (F685 and F695) are reduced by 20–30% (assuming no change in the PS I emission band). Thermoluminescence data with thylakoids show the absence of the S2Q−A and S2Q−B bands in the mutant. Herbicide 14C-terbutryn binding measurements, also with thylakoids, show that the QB niche of the mutant is significantly modified, at least 7–8 fold increased terbutryn dissociation constant is shown (220 nM in the mutant versus 29 nM in the wild type); the PS II sensitivity to bicarbonate-reversible formate inhibition is reduced by 5 fold in the mutant, although the formate/bicarbonate binding site still exists in the mutant. This suggests that D1-R269 must play some role in the binding niche of bicarbonate. On the basis of the above observations, we conclude that the D1-R269G mutation has not only altered the structure and function of PS II (QB niche being abnormal), but may also have a decreased net excitation energy transfer from the PS II core to the reaction center and/or an increased number of inactivated reaction center II. We also discuss a possible scenario for these effects using a recently constructed three dimensional model of the PS II reaction center.
Chloroacetates as inhibitors of photosystem II: Effects on electron acceptor side
1997, Journal of Photochemistry and Photobiology B: BiologyThe two-electron gate of Photosystem II (PSII) is known to function by transferring electrons from the reduced one electron acceptor QA− (a bound plastosemiquinone) to the oxidized two-electron acceptor QB (a bound plastoquinone), and then again from QA− to the singly reduced QB−, producing plastoquinol QBH2. In this article, we have three chloroacetates (monochloroacetate, MCA; dichloroacetate, DCA; and trichloroacetate, TCA), having different geometry and hydrophobicity, to probe the binding environment of the two-electron gate in spinach thylakoids. We first established that these chloroacetates up to 100 mM act specifically in the QAQB region by monitoring partial reactions of PSII as well as PSI, measuring thermoluminescence, specific for recombination reactions between the donor and acceptor sides of PSII, and studying chlorophyll (Chl) a fluorescence decay in the micro to millisecond region, specific for electron flow from QA− to QB. Further, the site of action was located on the D1–D2 protein through observations on the differential sensitivity of chloroacetates on specific D1–D2 mutants of the cyanobacterium Synechocystis sp. PCC 6803.
Detailed measurements were then done to characterized the effect of chloroacetates on QAQB reactions. Data on the [QA−] decay kinetics led to the following observations: (1) chloroacetates (and acetate) not only increase the time constant of electron flow from QA− to QB (QB−), but increase the equilibrium [QA−] both after flash 1 and 2, and the degree of these effects (lowest to highest) is correlated with the geometry (increased number of chlorine moiety) and increase hydrophobicity of these inhibitors: the hierarchy is: acetate < TCA. (2) In comparison with flash 1, data after flash 2 (at pH 6) show relatively larger increases in [QA−] equilibrium with DCA and TCA. At pH 7.5 however, flash 1 effects were larger than flash 2 effects with all chloroacetates. (3) Bicarbonate reverses the inhibitory effect on QA− to QA (QB−) reactions also in a differentimanner; the hierarchy for the most reversible (or least irrversible) is: acetate ⪆ MCA > DCA ⪢ TCA (4) The pH dependence of the inhibitory effects on QA− to QB (QB−)are: te MCA and DCA effects are larger at pH6 than at pH 7.5, but the TCA effects are higher at pH 7.5 than at pH 6. The above results, taken together with those in the literature, are in agreement with a picture of the QAFeQB niche in the D1–D2 protein of PSII where quinones, herbicides, chloroacetates, formate as well as bicarbonate bind, but differently with different overlapping sites. Chloroacetates show effects on the two-electron gate that place them ‘in between’ the herbicides and the bicarbonate-reversible formate.
Activation of 1-aminocyclopropane-1-carboxylate oxidase by carbon dioxide
1993, Biochemical and Biophysical Research Communications1-Aminocyclopropane-1-carboxylate (ACC) oxidase requires CO2/HCO3− as an essential activator for its activity. Taking advantage that the equilibrium concentrations of CO2 and HCO3− vary with pH and that the interconversions of CO2 and HCO3− arc slower at low temperature, we identified CO2 rather than HCO3− as the active species involved in the activation process. Preincubation of the enzyme with a saturating concentration of CO2 resulted in increased activation of the enzyme when preincuhation pH was raised, indicating that CO2 reacted with an enzyme group having an alkaline pKa. It is suggested that the CO2 activation of ACC oxidase involves the formation of a carhamate. CO2 increases the Vmax of the reaction but decreases the affinity of the enzyme toward its substrate ACC. A plausible reaction scheme accounting br the CO2 activation process is presented.