This Article Abstract Full Text (PDF) All Versions of this Article: 139/3/1444    most recent pp.105.068437v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via ISI Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Clausen, J. Articles by Messinger, J. Search for Related Content PubMed PubMed Citation Articles by Clausen, J. Articles by Messinger, J. Agricola Articles by Clausen, J. Articles by Messinger, J.

BIOENERGETICS AND PHOTOSYNTHESIS

Evidence That Bicarbonate Is Not the Substrate in Photosynthetic Oxygen Evolution¹

Abteilung Biophysik, Universität Osnabrück, 49069 Osnabrueck, Germany (J.C., W.J.); and Max-Planck-Institut für Bioanorganische Chemie, 45470 Muelheim an der Ruhr, Germany (K.B., J.M.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO₂ is an intermediate product of oxygen production in addition to O₂. In this work, we investigated this possible role of exchangeable HCO₃^– in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn₄Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO₂. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO₂ pressure over ambient conditions did not affect the UV transient, whereas the S₃ S₄ S₀ transition was half-inhibited by raising the O₂ pressure only 10-fold over ambient, as previously established. (2) The flash-induced O₂ and CO₂ production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H₂¹⁸O enrichment. Light flashes that revealed the known oscillatory O₂ release failed to produce any oscillatory CO₂ signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO₂ an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.

Bicarbonate has been clearly shown to bind at the non-heme iron of the acceptor side of PSII (Ferreira et al., 2004) and to be of functional relevance for electron transfer at this position (for a recent review, see van Rensen, 2002). In addition, there is evidence that bicarbonate also affects oxygen evolution directly. PSII of green algae, for example, is inhibited if bicarbonate is removed from the reaction mixture and substituted for by formate (Mende and Wiessner, 1985). Bicarbonate is also reported (1) to stabilize the OEC (for review, see Klimov and Baranov, 2001), (2) to promote the photoassembly of the Mn₄Ca complex (for review, see Ananyev et al., 2001), and (3) to interfere with the damping of the oscillatory pattern of oxygen evolution (Stemler et al., 1974). Finally, in the currently most complete structural model of PSII at 3.5 Å resolution (Ferreira et al., 2004), (bi)carbonate is presented as a ligand of the Mn₄Ca complex.

It seems generally accepted, however, that water serves as the electron donor for the production of reduced organic compounds by cyanobacteria and plants, and that dioxygen, the side product which is pivotal for aerobic life, is derived from water. Alternative concepts like Otto Warburg's (Warburg and Krippahl, 1958; Warburg et al., 1965), who proposed that "activated CO₂" is converted to an aldehyde during oxygen evolution (for a historical survey, see Stemler, 2002), have been discarded mainly because of mass-spectrometric results that show that in PSII preparations the isotopic composition of liberated dioxygen agrees well with the one of the surrounding water even after relatively short mixing times with H₂¹⁸O (Radmer and Ollinger, 1980; Bader et al., 1987; Messinger et al., 1995; Hillier et al., 1998; Hillier and Wydrzynski, 2000). Strictly speaking, however, these data are still compatible with a catalytic role of bicarbonate if the equilibrium between water, CO₂, and bicarbonate is rapidly installed, as mediated, for example, by a carbonic anhydrase (CA; Silverman and Tu, 1975; Tu and Silverman, 1975). An intrinsic CA activity is indeed linked to the core and/or extrinsic proteins of PSII (Dai et al., 2001; Stemler, 2002; Villarejo et al., 2002; Khristin et al., 2004). The above-cited mass-spectrometric experiments were not designed to specifically address the bicarbonate question, and therefore the CA activity was not assessed in detail. This leaves room for speculations about a possible catalytic role of bicarbonate during oxygen evolution.

Such a catalytic role for bicarbonate was first proposed by Helmut Metzner on thermodynamic grounds (Metzner, 1978). The main idea was that a one-electron oxidation of bicarbonate (HCO₃^–) is energetically possible for P680⁺, while a one-electron abstraction from water requires far too high oxidizing potentials. Therefore, he assumed that water is delivered to the OEC in a CO₂-bound form, i.e. as bicarbonate. This scheme requires that, in addition to O₂, CO₂ is a product. The formed CO₂ is proposed to be rapidly rehydrated to bicarbonate and therefore to act catalytically. Using today's knowledge about photosynthetic water oxidation, Metzner's idea can be reformulated in a way that appears consistent with presently published data:

(1)

(2)

(3)

wherein h stands for photon-driven processes. In this sequence reaction, (1) describes a series of four light-driven partial reactions involving two bicarbonate molecules (substrate) that are exchangeably bound to the OEC in the S₀ through S₃ states of the OEC. During the S₄ S₀ transition, O₂ and 2 CO₂ are formed and initially bound to the Mn₄Ca complex. In the next step they need to be released into the medium to allow another reaction cycle to occur (heterogeneous catalysis).

On the basis of this hypothetical reaction mechanism, two clear predictions can be made. (1) It can be expected that a high partial pressure of either O₂ or CO₂ suppresses oxygen production (this prediction is explained in more detail in the "Discussion"). (2) Because the reaction equilibrium of reaction 2 (K = [CO₂]/[H₂CO₃] = 600; Cotton and Wilkinson 1974) lies to 99.8% on the side of CO₂, a considerable amount of newly formed CO₂ should, at least transiently, escape together with O₂ into solution and become detectable in time-resolved experiments. In contrast, in the generally accepted view of photosynthetic oxygen evolution, with water as sole substrate, no CO₂ evolution and therefore also no inhibition by a high partial pressure of CO₂ are expected.

Given the described remaining chance for a catalytic role of bicarbonate as immediate substrate for photosynthetic oxygen production, we decided to test the modified Metzner model by two complementary approaches.

The stepped progression of the OEC was monitored by UV spectrophotometry under high backpressure of CO₂. In contrast to results from a previous study, where the oxygen-evolving S₄-to-S₀ transition was half-suppressed by only 10-fold elevated oxygen pressure (Clausen and Junge, 2004), the 3,000-fold increase of the CO₂ pressure did not affect the normal turnover of the OEC.

Measurements by membrane inlet mass spectrometry (MIMS), under H₂¹⁸O enrichment, and at low bicarbonate/CO₂ concentration revealed the normal oscillation of O₂ release, but they gave no evidence, under parallel detection, for any CO₂ release in response to a series of light flashes.

Both results practically exclude that CO₂ is liberated during oxygen evolution and thereby also show that bicarbonate is not an immediate/catalytic substrate as postulated by Metzner. Bicarbonate may, however, be a firmly bound cofactor.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Flash-Spectrometric Experiments

Figure 1 shows UV-absorption transients (wavelength 360 nm) of dark-adapted PSII-core particles from Synechocystis in response to series of short laser flashes given to dark-adapted samples. These transients are composites: They reflect redox reactions of the OEC and of the intrinsic and exogenous electron acceptors (see Lavergne, 1991; Dekker, 1992; Bögershausen and Junge, 1995; Karge et al., 1997; Clausen et al., 2001). With the catalytic center synchronized mainly in its first oxidation state, S₁, before starting the series of flashes, oxygen evolution occurs only after excitation with three flashes of light in a row, i.e. after reaching S₄. From there on it oscillates with period of four as a function of the flash number. Figure 1A shows an overlay of two UV transients. The trace shown in black represents the typical oscillation under atmospheric oxygen pressure (1 bar air, i.e. 0.21 bar O₂, pH 6.7, 20°C). On the first two flashes (S₁ S₂, S₂ S₃) the very rapid (here not resolved) rise of absorption is followed by a slower relaxation of smaller extent, so that each flash gives rise to a positive step. These steps represent oxidations of the OEC (Lavergne, 1991; Dekker, 1992; Bögershausen and Junge, 1995; Karge et al., 1997; Clausen et al., 2001). After the third flash (inducing the transition S₃ S₄ X S₀), these two positive steps are reverted by a negative transient with a typical half-decay time of 1.3 ms. It reflects the reduction of the Mn₄Ca complex that occurs concomitant with the liberation of dioxygen (see Clausen and Junge, 2004; Clausen et al., 2004) and sets the OEC back into the S₀ state. On the fourth flash (S₀ S₁) there is no positive step at 360 nm (Lavergne, 1991), and on the fifth flash a new cycle starts (S₁ S₂) with another positive jump. If the concentration of the product, oxygen, is increased to 20 bar (Fig. 1A, gray trace), then the magnitude of the negative jump decreases because the backpressure of O₂ stabilizes a so far ill-defined intermediate X of the transition S₃ S₄ X S₀ (for details, see Clausen and Junge, 2004).

View larger version (29K): [in this window] [in a new window]   Figure 1. UV absorption transients (360 nm) of dark-adapted PSII-core complexes (normalized to 9 µM Chl, pH 6.6 to 6.7) obtained by the excitation with five short laser flashes (arrows). A, The black trace was obtained after preincubation with 1 bar air [i.e. 0.21 bar p(O2)], while the gray trace shows the data that were obtained after preincubation with 20 bar pure O2. The transients were recorded with 50 µs/point and three experiments were averaged. B, The black trace represents again the air control (four transients averaged), while the gray trace gives the data obtained under 1.1 bar carbon dioxide (two transients averaged). These transients were recorded with a time constant of 100 µs/point.

The lack of any effect of high CO₂ pressure on oxygen production was corroborated by standard polarographic measurements of the rate of oxygen evolution under continuous illumination. The presence of 50 mM NaHCO₃ and 10 mM Na₂CO₃ in the reaction mixture yielded the same rate as observed in control samples without added NaHCO₃ and Na₂CO₃. There was neither a stimulatory nor an inhibitory effect of the greatly elevated CO₂ concentration (42.5 mM; Henry constant 38.66 mM bar^–1 at 20°C) on PSII turnover under steady illumination. This shows that the bicarbonate binding site of the OEC, if present, is already saturated by bicarbonate concentrations formed by ambient amounts of dissolved CO₂.

MIMS

Figure 2A shows mass-spectrometric signals resulting from the excitation of dark-adapted spinach (Spinacia oleracea) thylakoids with a series of 12 Xenon (Xe) flashes. They were recorded by MIMS using an isotope-ratio mass spectrometer that was set up to simultaneously measure O₂ and CO₂ at m/z = 32, 34, and 36, and 44, 46, and 48, respectively. For all three mass peaks of each gas, similar results were obtained. For clarity, Figure 2A shows only signals of the singly labeled species (mass 34 and 46, respectively). Before the measurements, the PSII samples (20 µM chlorophyll [Chl]/mL at pH 6.8) were mixed with approximately 15% H₂¹⁸O (final concentration) and then degassed in the sample chamber under rapid stirring to about 1.4 µM CO₂ (approximately 7% of the initial air-saturated value of 21 µM at 10°C) in order to achieve nearly constant baselines. A preflash and subsequent dark adaptation are used to convert all PSII centers of a given sample into the S₁Y_D^ox state. This procedure strongly reduces the S state decay between the required long dark times of 20 s between the flashes because it preoxidizes the endogenous electron donor Y_D that normally causes the fast phase of S₂ and S₃ state decay. The preflashed thylakoids are then illuminated with a train of 12 Xe flashes (approximately 3 µs full width at half maximum [FWHM]). The data in Figure 2A show that O₂ is evolved by a significant number of PSII complexes with high efficiency even at this low dissolved CO₂/HCO₃^– concentration. This is evident from (1) the clear period four oscillation of the O₂ signals, which can be well described with miss and double hit parameters of 14.3% and 3.8%, respectively, and 100% S₁ state dark population, and (2) that from the sum of the absolute amplitudes of the first 12 flashes a number of approximately 700 Chl per active PSII reaction center can be calculated. In contrast, the simultaneously recorded CO₂ trace does not show any flash-induced signals. In this regard it is important to note that the sensitivities of the two Fay cups are identical, and that the permeability of the silicone membrane for CO₂ is about 6 times greater than for O₂.

View larger version (17K): [in this window] [in a new window]   Figure 2. A, Flash-induced 18O16O (mass 34; solid line) and 12C18O16O (mass 46; dashed line) evolution of spinach thylakoid samples. A series of 12 saturating Xe flashes with intermittent dark times of 20 s was given at 10°C and pH 6.8. The H218O enrichment was 15% and the Chl concentration 0.02 mg Chl/mL. The signal induced by the third of the flash corresponds to 4.8 pmol O2. For better comparison, a monoexponentially decaying baseline was subtracted from the CO2 signal, which had a significantly higher starting value. B, Change in 12C18O2 concentration as a function of time after the injection of 28 µL of H218O (95% initial; 15% final enrichment). The concentration of spinach thylakoids was 0.20 mg Chl/mL (a), 0.02 mg Chl/mL (b), and 0.00 mg Chl/mL (c). Other conditions are as in Figure 2A.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

Another possible function of bicarbonate is that of a cofactor of photosynthetic water oxidation. In contrast to substrates, cofactors may be relatively tightly associated with the enzyme. Such a possible cofactor role of bicarbonate in photosynthetic water oxidation, which may, for example, involve (1) a function in the proton relay network, (2) a structural role, and/or even (3) provide a binding site for one substrate water molecule, cannot be analyzed by our current approaches.

In the first series of experiments, the reduction of the Mn₄Ca complex during the oxygen-evolving reaction step was followed by monitoring absorption transients in the near UV. For the case that bicarbonate is the immediate substrate of the OEC, it is expected that greatly increased partial pressure of CO₂ should suppress this step. This product inhibition was previously observed for O₂ using moderately increased partial pressures. Nevertheless, for bicarbonate this expectation may not be immediately obvious on the basis of the simple reaction scheme presented in Equations 1 to 3 because increased CO₂ pressure also causes proportionally increased concentrations of HCO₃^– via the reactions shown in Equation 2, which in principle can counterbalance the effect on the product side (Eq. 1). However, because we are concerned with a light-driven heterogeneous catalysis, this cancellation of effects cannot happen. In order to illustrate this important point, we shall concentrate only on the final steps of the postulated reaction sequence that are summarized in more detail in Equation 4:

(4)

Herein, BCA stands for bicarbonate, S₃ and S₄ for the catalytic center (OEC plus Y_Z) in its third and fourth oxidation states, X for an ill-characterized intermediate, and h for the (usually third) photon that initiates oxygen liberation. Protons are omitted for simplicity. The existence of intermediate X was established elsewhere (Clausen and Junge, 2004), but it can be omitted in the following argument. Equation 4 accounts for three important properties. (1) The right reaction starting from S₄ is heterogeneous in that a solid component cleaves off two soluble components, O₂ and CO₂. Its equilibrium is therefore shifted to the left if the partial pressure of either product is increased. (2) The middle reaction (S₃ to S₄) is photochemical and does not proceed without the extra driving force provided by a quantum of light. Therefore, the equilibrium of the left reaction (binding of bicarbonate to the S₃ state) does not directly bear on the equilibrium of the right because both parts are shielded from each other by the photochemical step. In this particular case, there is additionally good reason to suppose saturation of bicarbonate binding, because (1) the mass-spectrometric experiments show significant O₂ evolution at greatly diminished bicarbonate concentrations and (2) because addition of bicarbonate to PSII suspensions did not increase the rate of oxygen evolution (see above). These arguments show that photosynthetic oxygen evolution should be very sensitive to increased pressure of CO₂, if bicarbonate is the substrate. However, the data of Figure 1B show no inhibition of the S₃ S₄ S₀ transition in cyanobacterial core complexes by very large (x3,000) CO₂ overpressure. This result excludes all hypothetical reaction schemes involving exchangeable bicarbonate/CO₂. This conclusion is further corroborated by our MIMS experiments, which give no evidence for any oscillating CO₂ release down to a level of 2% below the one of the oscillating oxygen production.

A weak point of previous time-resolved mass-spectrometric experiments was that they did not determine the CA activity in the samples (see introduction). We show here that the CA activity of spinach thylakoids can be determined from the decay of the mass-spectroscopic signal at a normalized mass of 48 (¹²C¹⁸O¹⁸O; Fig. 2B). From the data of Table I it can be estimated that under the conditions employed in the previous water exchange experiments (thylakoid concentrations of e.g. 0.25 mg Chl/mL; Messinger et al., 1995) a comparatively long time (>30 s) is required for reaching the isotopic equilibrium between CO₂/HCO₃^– and water. This is in contrast to the rapid isotopic equilibrium in water, which is reached by physical mixing in the microsecond time scale (Messinger et al., 1995; Hillier et al., 1998; Hillier and Wydrzynski, 2000; Hendry and Wydrzynski, 2002). The isotopic equilibration of water is essentially unaffected by the subsequent equilibration with the CO₂ species because of the far smaller concentration of the latter species. Since at 10°C the ¹⁸O label quantitatively appears in flash-induced oxygen signals within about 3 s (k_slow = 2.2 s^–1; Messinger et al., 1995), the combination of these two data sets supports also strongly the view that water, and not bicarbonate, is directly oxidized by PSII. The function of the observed CA activity of PSII and of thylakoid membranes as a whole remains to be elucidated (see Table I and Fig. 2B; for review, see Stemler, 1998, 2002). It has been suggested that the lumenal CA of thylakoids may play a role in the formation of a proton gradient across the thylakoid membrane in the dark in order to support ATP synthesis (van Hunnik and Sultemeyer, 2002). CA may also be involved in regulating the pH of the lumen similar to its known function in blood.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The results presented in this article refute any direct role of exchangeable bicarbonate/CO₂ as substrate/intermediate of photosynthetic oxygen evolution. Instead they corroborate the view that water is the direct source of electrons for the production of carbohydrates and, likewise, the source of the oxygen we breathe.

The same results do not rule out the involvement of firmly bound or sequestered bicarbonate in water oxidation. It remains conceivable that bound HCO₃^– may (1) be part of a deprotonation pathway, (2) alter redox properties of the Mn₄Ca complex, (3) stabilize the metal-cluster as a ligand to manganese and/or calcium (Klimov et al., 1995), or (4) provide a binding site for substrate water.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Flash-spectrometric experiments were carried out with oxygen-evolving PSII-core particles prepared from modified wild-type cells of Synechocystis sp. PCC6803 as described elsewhere (Clausen et al., 2001). Aliquots were suspended in standard medium: 1 M Suc, 25 mM CaCl₂, 10 mM NaCl, 1 M Gly betaine, 50 mM MES (1 M MES for experiments under 1.1 bar CO₂-pressure), pH 6.6 to 6.7. The temperature was 20°C ± 0.5°C.

The home-built optical cell with fused sapphire windows sustained pressures up to 30 bar (Clausen et al., 2001). The standard reaction medium was pre-equilibrated with purified oxygen or carbon-dioxide gas under the desired partial pressure for at least 40 min. It was then filled into the optical cell (optical pathlength = 1 cm) and re-equilibrated with the pressurized gas phase for another 10 min. A small aliquot (typically 50 µL) of dark-adapted PSII particles from the concentrated stock was then injected into the cell, which was filled with roughly 3 mL of liquid and 20 mL of gas to give final concentrations of 7 to 15 µM Chl and 0.06% (w/v) N-dodecyl--D-maltoside. The mixture was kept in darkness and equilibrated at the chosen pressure for another 20 min under gentle stirring. After addition of the electron acceptor (200 µM 2,5-dichloro-p-benzoquinone), the sample was excited under pressure with a group of five flashes from a Q-switched Nd-YAG laser (wavelength 532 nm, duration 6 ns FWHM, saturating energy, 100 ms between flashes) and the absorption transients were recorded at a wavelength of 360 nm. This wavelength was chosen to reveal charge transfer bands of the Mn₄Ca complex with minimal contributions from Tyr (Dekker et al., 1984; Schatz and van Gorkom, 1985; Weiss and Renger, 1986; Gerken et al., 1989; Haumann et al., 1999). Data were recorded at an analog bandwidth of 10 kHz and digitized at 50 µs or 100 µs per bin. Control samples were prepared in the same manner, except that they were stirred for 20 min under 1 bar air (0.21 bar oxygen).

MIMS experiments were carried out with spinach (Spinacia oleracea) thylakoids, which were suspended in a buffer containing 5 mM CaCl₂, 50 mM MES (pH 6.8/10°C), 5 mM MgCl₂, 15 mM NaCl, and 400 mM Suc to give a final concentration of 0.02 mg Chl/mL. The experiments were carried out in a home-built cell similar to that described by Messinger et al. (1995), but with 150 µL sample volume and an improved mixing system. The gas inlet system was formed by a silicone membrane (MEM-213) that rests on a porous plastic support, which together separated the aqueous sample from the vacuum (3 x 10^–8 bar) of the isotope ratio mass spectrometer (ThermoFinnigan Delta^plusXP), which was equipped with seven Fay cups for the simultaneous detection of masses 32, 34, 36, 40, 44, 46, and 48. No artificial electron acceptors were added. Dark-adapted samples were excited with a Xe flash lamp with approximately 3 µs FWHM. The individual flashes were separated by dark intervals of 20 s. The calibration of the CO₂ and O₂ signals was performed by injecting various volumes of air-saturated water samples (21 µM CO₂ and 351 µM O₂ at 10°C) into the sample chamber filled with degassed buffer.

	ACKNOWLEDGMENTS

 
We thank Hella Kenneweg (Osnabrück) and Birgit Nöring (Mülheim/Ruhr) for excellent technical assistance, Holger Heine (Osnabrück) for advise on the construction of the pressure cell, Prof. Rick Debus (Riverside) for the cooperation on Synechocystis, Drs. Ralf Ahlbrink and Armen Mulkidjanian (both Osnabrück) for fruitful discussions and help, and Govindjee (Urbana) and the referees for helpful comments on the manuscript.

Received July 15, 2005; returned for revision September 2, 2005; accepted September 2, 2005.

	FOOTNOTES

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. Ju97/15–3 and Me1623/2–3), the Fonds der Chemischen Industrie (W.J.), the Max-Planck Society (J.M.), and the Land Niedersachsen (W.J.).

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) are: Wolfgang Junge (junge{at}uos.de) and Johannes Messinger (messinger{at}mpi-muelheim.mpg.de).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.068437.

^* Corresponding author; e-mail junge{at}uos.de; fax 49–541–969–2262.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
Ananyev GM, Zaltsman L, Vasko C, Dismukes GC (2001) The inorganic biochemistry of photosynthetic oxygen evolution/water oxidation. Biochim Biophys Acta 1503: 52–68[Medline]

Bader KP, Thibault P, Schmid GH (1987) Study on the properties of the S₃-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea. Biochim Biophys Acta 893: 564–571

Biesiadka J, Loll B, Kern J, Irrgang KD, Zouni A (2004) Crystal structure of cyanobacterial photosystem II at 3.2 angstrom resolution: a closer look at the Mn-cluster. Phys Chem Chem Phys 6: 4733–4736[CrossRef][ISI]

Bögershausen O, Junge W (1995) Rapid proton transfer under flashing light at both functional sides of dark adapted photosystem II core particles. Biochim Biophys Acta 1230: 177–185[CrossRef]

Cinco RM, Robblee JH, Messinger J, Fernandez C, Holman KLM, Sauer K, Yachandra VK (2004) Orientation of calcium in the Mn4Ca cluster of the oxygen-evolving complex determined using polarized strontium EXAFS of photosystem II membranes. Biochemistry 43: 13271–13282[Medline]

Clausen J, Debus RJ, Junge W (2004) Time-resolved oxygen production by PSII: chasing chemical intermediates. Biochim Biophys Acta 1655: 184–194[Medline]

Clausen J, Junge W (2004) Detection of an intermediate of photosynthetic water oxidation. Nature 430: 480–483[CrossRef][Medline]

Clausen J, Winkler S, Hays AMA, Hundelt M, Debus RJ, Junge W (2001) Photosynthetic water oxidation: Mutations of D1-Glu189K, R and Q of Synechocystis sp. PCC6803 are without any influence on electron transfer rates at the donor side of photosystem II. Biochim Biophys Acta 1506: 224–235[Medline]

Cotton FA, Wilkinson G (1974) Anorganische Chemie. Verlag Chemie, Weinheim, Germany, pp 1–1235

Dai XB, Yu Y, Zhang RX, Yu XJ, He PM, Xu CH (2001) Relationship among photosystem II carbonic anhydrase, extrinsic polypeptides and manganese cluster. Chin Sci Bull 46: 406–409

Dekker JP (1992) Optical studies on the oxygen-evolving complex of photosystem II. In VL Pecoraro, ed, Manganese Redox Enzymes, Ed 1. VCH Publishers, New York, pp 85–104

Dekker JP, van Gorkom HJ, Brok M, Ouwehand L (1984) Optical characterization of photosystem II electron donors. Biochim Biophys Acta 764: 301–309[CrossRef]

Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Architecture of the photosynthetic oxygen-evolving center. Science 303: 1831–1838[Abstract/Free Full Text]

Gerken S, Dekker JP, Schlodder E, Witt HT (1989) Studies on the multiphasic charge recombination between chlorophyll A_II⁺ (P-680⁺) and plastoquinone Q_A^– in Photosystem II complexes. Ultraviolet difference spectrum of Chl-a_II⁺/Chl-a_II. Biochim Biophys Acta 977: 52–61

Haumann M, Mulkidjanian A, Junge W (1999) Tyrosine-Z in oxygen evolving photosystem II: a hydrogen-bonded tyrosinate. Biochemistry 38: 1258–1267[CrossRef][Medline]

Hendry G, Wydrzynski T (2002) The two substrate-water molecules are already bound to the oxygen-evolving complex in the S2 state of photosystem II. Biochemistry 41: 13328–13334[Medline]

Hillier W, Messinger J, Wydrzynski T (1998) Kinetic determination of the fast exchanging substrate water molecule in the S₃ state of photosystem II. Biochemistry 37: 16908–16914[CrossRef][Medline]

Hillier W, Wydrzynski T (2000) The affinities for the two substrate water binding sites in the O(2) evolving complex of photosystem II vary independently during S-state turnover. Biochemistry 39: 4399–4405[CrossRef][Medline]

Kamiya N, Shen JR (2003) Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-angstrom resolution. Proc Natl Acad Sci USA 100: 98–103[Abstract/Free Full Text]

Karge M, Irrgang KD, Renger G (1997) Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H₂O or D₂O. Biochemistry 36: 8904–8913[CrossRef][Medline]

Khristin MS, Ignatova LK, Rudenko NN, Ivanov BN, Klimov VV (2004) Photosystem II associated carbonic anhydrase activity in higher plants is situated in core complex. FEBS Lett 577: 305–308[Medline]

Klimov VV, Allakhverdiev SI, Feyziev YM, Baranov SV (1995) Bicarbonate requirement for the donor side of photosystem II. FEBS Lett 363: 251–255[Medline]

Klimov VV, Baranov SV (2001) Bicarbonate requirement for the water-oxidizing complex of photosystem II. Biochim Biophys Acta 1503: 187–196[Medline]

Kok B, Forbush B, McGloin M (1970) Cooperation of charges in photosynthetic O₂ evolution. I. A linear four-step mechanism. Photochem Photobiol 11: 457–475[ISI][Medline]

Lavergne J (1991) Improved UV-visible spectra of the S-transitions in the photosynthetic oxygen-evolving system. Biochim Biophys Acta 1060: 175–188

Mende D, Wiessner W (1985) Bicarbonate in vivo requirement of photosystem II in the green alga Chlamydobotrys stellata. J Plant Physiol 118: 259–266

Messinger J (2004) Biophysical studies of Photosystem II and related model systems. Phys Chem Chem Phys 6: E11–E12[CrossRef]

Messinger J, Badger M, Wydrzynski T (1995) Detection of one slowly exchanging substrate water molecule in the S₃ state of photosystem II. Proc Natl Acad Sci USA 92: 3209–3213[Abstract/Free Full Text]

Metzner H (1978) Photosynthetic Oxygen Evolution. Academic Press, London

Mills GA, Urey HC (1940) The kinetics of isotopic exchange between carbon dioxide, bicarbonate ion, carbonate ion and water. J Am Chem Soc 62: 1019–1025[CrossRef]

Radmer R, Ollinger O (1980) Isotopic composition of photosynthetic O₂ flash yields in the presence of H₂¹⁸O and HC¹⁸O₃^–. FEBS Lett 110: 57–60

Robblee JH, Messinger J, Cinco RM, McFarlane KL, Fernandez C, Pizarro SA, Sauer K, Yachandra VK (2002) The Mn cluster in the S-0 state of the oxygen-evolving complex of photosystem II studied by EXAFS spectroscopy: Are there three di-mu-oxo-bridged Mn-2 moieties in the tetranuclear Mn complex? J Am Chem Soc 124: 7459–7471[CrossRef][ISI][Medline]

Schatz GH, van Gorkom HJ (1985) Absorbance difference spectra upon charge transfer to secondary donors and acceptors in photosystem II. Biochim Biophys Acta 810: 283–294

Silverman DN, Tu CK (1975) Buffer dependence of carbonic-anhydrase catalyzed oxygen-18 exchange at equilibrium. J Am Chem Soc 97: 2263–2269[CrossRef]

Stemler A, Babcock GT, Govindjee (1974) The effect of bicarbonate on photosynthetic oxygen evolution in flashing light in chloroplast fragments. Proc Natl Acad Sci USA 71: 4679–4683[Abstract/Free Full Text]

Stemler AJ (1998) Bicarbonate and photosynthetic oxygen evolution: an unwelcome legacy of Otto Warburg. Indian J Exp Biol 36: 841–848

Stemler AJ (2002) The bicarbonate effect, oxygen evolution, and the shadow of Otto Warburg. Photosynth Res 73: 177–183[Medline]

Tu CK, Silverman DN (1975) Kinetics of exchange of oxygen between carbon-dioxide and carbonate in aqueous-solution. J Phys Chem 79: 1647–1651[CrossRef]

van Hunnik E, Sultemeyer D (2002) A possible role for carbonic anhydrase in the lumen of chloroplast thylakoids in green algae. Funct Plant Biol 29: 243–249[CrossRef]

van Rensen JJS (2002) Role of bicarbonate at the acceptor side of Photosystem II. Photosynth Res 73: 185–192[Medline]

Villarejo A, Shutova T, Moskvin O, Forssen M, Klimov VV, Samuelsson G (2002) A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution. EMBO J 21: 1930–1938[CrossRef][ISI][Medline]

Warburg O, Krippahl G (1958) Hill-Reaktionen. Z Naturforsch B 13B: 509–514

Warburg O, Krippahl G, Jetschma C (1965) Widerlegung der Photolyse des Wassers und Beweis der Photolyse der Kohlensäure nach Versuchen mit lebender Chlorella und den Hill-Reagentien Nitrat und K₃Fe(Cn)₆. Z Naturforsch B B20: 993–996

Weiss W, Renger G (1986) Studies on the nature of the water-oxidizing enzyme. II. On the functional connection between the reaction-center complex and the water-oxidizing system Y in Photosystem II. Biochim Biophys Acta 850: 173–183

Zouni A, Witt HT, Kern J, Fromme P, Krauss N, Saenger W, Orth P (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409: 739–743[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 139/3/1444    most recent pp.105.068437v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via ISI Web of Science (12) Citing Articles via Google Scholar Google Scholar Articles by Clausen, J. Articles by Messinger, J. Search for Related Content PubMed PubMed Citation Articles by Clausen, J. Articles by Messinger, J. Agricola Articles by Clausen, J. Articles by Messinger, J.