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J. Biol. Chem., Vol. 276, Issue 26, 23450-23455, June 29, 2001

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Passive Entry of CO₂ and Its Energy-dependent Intracellular Conversion to HCO in Cyanobacteria Are Driven by a Photosystem I-generated µH^+*

Dan Tchernov, Yael Helman, Nir Keren, Boaz Luz, Itzhak Ohad, Leonora Reinhold, Teruo Ogawa^§, and Aaron Kaplan^¶

From the  Faculty of Science and Mathematics and The Minerva Center for Photosynthesis under Stress, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel and the ^§ Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan

Received for publication, March 5, 2001, and in revised form, April 5, 2001

	ABSTRACT

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CO₂ entry into Synechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO₂ uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO. Dependence of CO₂ uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO₂ uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a µH⁺ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO₂ uptake. Under low light intensity, the rate of CO₂ uptake by a high-CO₂-requiring mutant of Synechococcus sp. PCC7942, at a CO₂ concentration below its threshold for CO₂ fixation, was higher than that of the wild type. At saturating light intensity, net CO₂ uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO₂ to HCO. These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO₂ to HCO.

	INTRODUCTION

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On illumination, many photosynthetic microorganisms maintain the concentration of dissolved CO₂ ([CO_2(dis)]) in their surrounding medium below that expected at chemical equilibrium with HCO (1-5). This displacement of [CO_2(dis)] from equilibrium can be observed in the absence of CO₂ fixation and is largely due to CO₂ uptake, intracellular conversion to HCO, and release of the latter into the medium (5, 6). The reverse phenomenon has been described in Synechococcus WH 7803 (7) and Nannochloropsis sp. (8, 9) where HCO uptake, internal conversion to CO₂, and efflux of the latter result in elevated [CO_2(dis)] in the medium.

HCO transport systems, in Cyanobacteria, are probably located at the cytoplasmic membrane and are believed to be driven by ATP either directly (10) or possibly indirectly (11-13). CO₂ uptake has been observed to result in HCO accumulation in the cytoplasm where [CO_2(dis)] is maintained below that expected at chemical equilibrium, and it has been inferred that a CA¹-like activity is involved in its uptake and intracellular conversion to HCO (6, 14-17). The location of the CA-like activity has not been identified and the mode of energization of the active HCO accumulation is not understood. Active transport of CO₂ across the plasmalemma has also been suggested (5, 18), but it is difficult to distinguish this from diffusion of CO₂ across the plasma membrane with subsequent energy-dependent conversion to HCO (6, 19). Passive entry of CO₂ across the membrane may occur via aquaporins (20), a possibility examined here by the application of a water channel blocker (WCB), p-chloromercuriphenylsulfonic acid. Use of this WCB prevented changes in cell volume and inactivation of PSI and PSII following osmotic stress in Synechococcus sp. strain PCC7942 (21).

Until recently, it was widely accepted that cyclic PSI activity plays the major role in energization of CO₂ uptake in Cyanobacteria (22, 23). Some recent observations appear to conflict with this conclusion. In the ndhD1/D2 mutant of Synechocystis sp. strain PCC6803 lacking components of NAD(P)H dehydrogenase (NDH-1), oxidation of P700 was depressed, but CO₂ uptake was only slightly affected (24). On the other hand, in the ndhD3/D4 mutant (25) and the ndhD3 mutant of Synechococcus sp. strain PCC7002 (26) CO₂ uptake was drastically depressed with only a small effect on P700 oxidation (i.e. PSI cyclic electron transport, ET). We have therefore reexamined the involvement of PSI in CO₂ uptake and suggest how the former data may be reconciled.

We have recently suggested a working hypothesis according to which CO₂ uptake by Cyanobacteria and its intracellular conversion to HCO may be energized by photosynthetic electron transport via the formation of alkaline domains on the stromal face of the thylakoid membrane (6). Catalyzed conversion of CO₂ to HCO in these domains would maintain an inward diffusion gradient for CO₂. Results presented are consistent with a prediction of this model, i.e. that CO₂ uptake would depend on a µH⁺ rather than on ATP hydrolysis.

	MATERIALS AND METHODS

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Synechococcus sp. PCC7942, Synechocystis PCC6803, and mutants thereof were grown on BG-11 medium (27) supplemented with 20 mM Hepes-NaOH, pH 8.0, and with 5 mM glucose in the case of the Synechocystis PCC 6803 mutant psaA/B (28). The cultures were aerated with either high or low CO₂ concentration (5% CO₂ in air or 1:1 mixture of air and CO₂-free air, respectively), at 30 °C and light intensity of 100 µmol photons m² s¹. The cells were harvested during the log phase of growth and resuspended in growth media.

Gas exchange measurements were performed with a membrane inlet mass spectrometer (Balzers QMG 421) as described earlier (7). Changes in the concentration of one gas affects the signal obtained for the others and, if not corrected for, may lead to erroneous interpretation of the results. Simultaneous measurements of argon and nitrogen concentrations were therefore used to correct for variations or drifts in the system due to biological formation or consumption of O₂ and CO₂, small changes in the rate of stirring or temperature, and in the gas consumption by the mass spectrometer. The latter was minimized by using silicon tubing with a small surface area (7). The cells were placed in a temperature-controlled chamber (2.8 ml) at 30 °C and illuminated with two optic fibers at the desired light intensity. The various chemicals supplied to the cell suspension were introduced via a special injection port.

Fig. 1 may serve as an example of the correct interpretation of curves obtained using the closed membrane introduction mass spectrometry chamber. Upon illumination of Synechococcus sp. strain PCC7942 cells the dissolved CO₂ concentration ([CO_2(dis)]) in the medium declined steeply even prior to the onset of net O₂ evolution (see Fig. 1, panels A and B). Note that the slope of the curve relating external [CO_2(dis)] to time (Panel B) cannot be taken as a direct indication of the initial rate of CO₂ removal by the cells because CO₂ is being formed continuously in the solution by net dehydration of HCO. Moreover, even though HCO concentration is virtually constant under the conditions of this experiment, net dehydration rate is not constant but rises because of the decline in CO₂ hydration rate as [CO_2(dis)] drops. Consequently, the further [CO_2(dis)] deviates from chemical equilibrium with HCO the lower the rate of CO₂ hydration, and therefore the higher the rate of net dehydration. At plateaus in the curve, where the CO₂ concentrations are relatively constant, the net rate of CO₂ uptake by the cells will be equal to the net rate of CO₂ formation by dehydration of HCO in the medium.

	RESULTS

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Displacement of [CO_2(dis)] from Chemical Equilibrium upon Illumination-- The extent of [CO_2(dis)] displacement from equilibrium was strongly affected by light intensity (Figs. 1 and 3). Raising the latter from 85 µmol photons m² s¹ (Fig. 1, panel B) to 750 µmol photons m² s¹ (panel C) led to an immediate drop in the ambient [CO_2(dis)] suggesting a higher rate of net CO₂ uptake. The rate of O₂ evolution also increased from 130 to 310 µmol O₂ mg¹ Chl h¹. Return to the light intensity of 85 µmol photons m² s¹ (panel D) caused the [CO_2(dis)] curve to rise again to a level higher than in the preceding light period at this intensity (compare panels B and D). The upward slope of the [CO_2(dis)] curve at high light intensity (Fig. 1, panel C) and the following downward slope at low light (panel D) most likely reflect changes in the light-driven ET rate via the photosystems due to adjustments in the efficiency of energy transfer from the phycobilisomes.² Upon darkening (Fig. 1, panel E), the [CO_2(dis)] rose rapidly to the equilibrium value as the net rate of CO₂ uptake fell below the dehydration rate. The [CO_2(dis)] frequently rose transiently above that expected at equilibrium probably due to formation of CO₂ from HCO in the intracellular C_i pool and leak of the former to the medium (2). Note that in the second half of the period of high illumination (panel C), the rate of O₂ evolution rose as the ambient concentration of CO₂ increased, i.e. slower net CO₂ uptake. Moreover, while the ambient [CO_2(dis)] declined (during the second half of panel D) indicating a rising rate of CO₂ uptake, the rate of O₂ evolution remained constant. If CO₂ fixation accounted for alterations in [CO_2(dis)], one would have expected that changes in the CO₂ uptake curve to be the mirror image of those in the O₂ evolution curve, but that is not the case. These data provide supporting evidence for the conclusion that CO₂ uptake does not solely reflect CO₂ fixation and may occur even in its absence (5, 6).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Changes in CO2 and O2 concentration in the medium surrounding high-CO2-grown Synechococcus PCC 7942 as affected by duration and intensity of illumination. The latter is given above the curves in µmol photons m2 s1. The cell suspension corresponded to 10 µg Chl/ml. Ci concentration was 1 mM, 30 °C.

Dependence of CO₂ Uptake on Light Intensity-- To distinguish between the effects of light intensity on CO₂ uptake and on CO₂ fixation, we used the high-CO₂-requiring mutant of Synechococcus PCC7942, IL-3 (32) which maintains the [CO_2(dis)] below CO₂/HCO equilibrium even at CO₂ concentrations lower than its threshold for net CO₂ fixation (6). High-CO₂-grown cells of Synechococcus PCC 7942 and of mutant IL-3 were exposed to a range of light intensities in the membrane introduction mass spectrometry chamber. The cells were provided with 1 mM C_i, sufficient to saturate photosynthesis in the case of the wild type but too low to enable CO₂-dependent O₂ evolution in the case of the mutant. At light intensities below 200 µmol photons m² s¹, CO₂ uptake by IL-3 was considerably faster than in the wild type (Fig. 2). At higher light intensities, the rates of net CO₂ uptake by the mutant and Synechococcus PCC7942 were similar (Fig. 2, inset). The rates of net CO₂ uptake declined when cells of Synechococcus or mutant IL-3 were exposed to light intensity higher than 600 µmol photons m² s¹ (Fig. 2, inset) probably due to photoinhibition.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   The effect of light intensity on the net rate of CO2 uptake by Synechococcus PCC 7942 and mutant IL-3. The inset provides the results obtained over the entire range of light intensities examined. Cell density corresponded to 4 µg Chl/ml, and Ci concentration was 1 mM, sufficient to saturate CO2 fixation by the wild type but below the threshold level required in the mutant. Temperature was 30 °C.

Inhibition of CO₂ Uptake by a WCB-- Addition of the WCB p-chloromercuriphenylsulfonic acid to a cell suspension of high-CO₂-grown Synechococcus PCC7942 resulted in severe, almost complete, inhibition of net CO₂ uptake by over 90% (as calculated from the CO₂ concentration at the plateau attained after the addition of the WCB, Fig. 3A). Photosynthetic O₂ evolution was also severely depressed. To distinguish between a direct effect of the WCB on CO₂ uptake and a possible indirect effect due to the decline in CO₂ fixation, we applied iodoacetamide (IAC) that completely inhibits CO₂ fixation (5, 6, 29-31). We also made use of the high-CO₂-requiring mutant of Synechococcus PCC7942, IL-3 in which the light-saturated rate of CO₂ uptake is similar to that of its wild type (Fig. 2) even at CO₂ concentrations lower than its threshold for net CO₂ fixation (6). In the presence of IAC the rate of net CO₂ uptake only declined by about 20% (Fig. 3B), although O₂ evolution was completely suppressed (not shown) providing further evidence that displacement of [CO_2(dis)] from equilibrium may occur irrespective of whether or not CO₂ is fixed. Addition of the WCB either to IAC-treated wild type or to IL-3 cells inhibited CO₂ uptake almost completely (Fig. 3B). The possibility that WCB inhibition of CO₂ uptake and fixation reflected severe unspecific damage to the cells was examined by raising the concentration of C_i in the medium. Normal photosynthetic rates (306 µmol O₂ evolved mg¹ Chl h¹) were observed when WCB-treated Synechococcus cells were supplemented with 20 mM C_i. These data indicate that a reduced availability of CO₂ to otherwise fully functional photosynthetic machinery led to the inhibition of CO₂ fixation by p-chloromercuriphenylsulfonic acid. Interestingly, Synechocystis PCC6803 is far less inhibited by the WCB than Synechococcus PCC7942 for a reason yet unknown.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   The effect of a water channel blocker on net CO2 uptake by Synechococcus PCC 7942 and its mutant IL-3. Cell density corresponded to 4 µg Chl/ml, and light intensity was 160 µmol photons m2 s1. Other conditions were as in Fig. 1. A, inhibition of net CO2 uptake following the addition of the water channel blocker (WCB) p-chloromercuriphenylsulfonic acid (21) to Synechococcus PCC 7942. B), rates of net CO2 uptake by Synechococcus PCC 7942 and mutant IL-3 following iodoacetamide (IAC, 3.3 mM provided in the dark 8 min prior to illumination) and of WCB treatments. The data are presented as a percentage of those exhibited by the wild type (270 µmol CO2 mg1 Chl h1).

Dependence of CO₂ Uptake on Photosynthetic Electron Transport via PSI-- The functional linkage between photosynthetic ET and CO₂ uptake was examined with the aid of electron acceptors, donors, and electron transfer inhibitors. In addition to Synechococcus PCC7942 we also examined Synechocystis PCC6803, which exhibits a similar displacement of [CO_2(dis)] from equilibrium upon illumination and where mutants impaired in PSI activity are available. As previously reported (33), inhibition of linear electron flow by DCMU abolished CO₂ uptake (Fig. 4A). However, addition of duroquinol that donates electrons to plastoquinol and reduces cytochrome b6f thus priming light-driven PSI electron flow, reestablished CO₂ uptake but not CO₂ fixation (completely inhibited by DCMU). These results provide further evidence that generation of CO₂/HCO disequilibrium is not compulsorily linked to CO₂ removal by the carboxylation reactions but does require PSI activity (Fig. 4A).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   The effect of electron acceptors, donors, transfer inhibitors, and mutations in components of the photosynthetic electron transport chain on the extent of the light-induced displacement of [CO2(dis)] from chemical equilibrium. A., inhibition by DCMU and alleviation of the inhibition by reduced duroquinone (DQH2). DCMU and DQH were added to final concentrations of 105 M and 2 × 103 M, respectively. The Synechococcus PCC 7942 cell density corresponded to 10 µg Chl/ml. B, inability of mutant psaA/B of Synechocystis PCC 6803 to displace [CO2(dis)] from equilibrium even after addition of 2,5-dimethyl-p-benzoquinone (DMBQ), which accepts electrons from PSII. Cell density corresponded to 10 µg Chl/ml. C, inversion of CO2/HCO disequilibrium after supply of DMBQ to Synechococcus PCC 7942 and return to equilibrium after supply of carbonic anhydrase (CA). Cell density corresponded to 15 µg Chl/ml. D, inversion of CO2/HCO disequilibrium after supply of methyl viologen (MV) to Synechococcus PCC 7942, abolition of disequilibrium in the dark, recreation of disequilibrium in a subsequent light period, and return to equilibrium after supply of CA. Cell density corresponded to 8 µg Chl/ml.

Direct evidence for the role of PSI in CO₂ uptake was obtained by use of a psaA/B mutant of Synechocystis PCC6803 that lacks a functional PSI (28). The mutant was unable to displace [CO_2(dis)] from equilibrium even when supplied with DMBQ, an efficient electron acceptor of PSII (Fig. 4B). The high DMBQ-dependent O₂ evolution (290 µmol O₂ mg¹ Chl h¹, Fig. 4B) indicated significant PSII activity in the mutant. This mutant obviously is neither able to fix CO₂ nor to perform cyclic electron flow in the light. Thus linear electron flow from PSII via plastoquinone to DMBQ, which does not involve PSI, is not sufficient to drive light-dependent CO₂ uptake.

Addition of the electron acceptors DMBQ or methyl viologen (MV) that draw electrons from PSII and PSI, respectively thus inhibiting cyclic electron flow via PSI, resulted in net CO₂ extrusion in Synechococcus. The [CO_2(dis)] at steady state was higher than expected at CO₂/HCO equilibrium (Fig. 4, C and D). Addition of CA to the medium reestablished the chemical equilibrium value (Fig. 4C). Similar results were obtained when MV was supplied to mutant M55 of Synechocystis PCC6803 in which ndhB, the encoding subunit II of NAD(P)H dehydrogenase NDH-1, was inactivated (not shown). Net CO₂ efflux of the magnitude observed in the presence of MV is highly likely to be the consequence of net HCO uptake followed by intracellular conversion to CO₂ and leak of the latter to the medium (7, 23, 34). These results suggest potential reversibility of the direction of the C_i cycling. The psaA/B mutant did not evolve CO₂ in the presence of DMBQ (Fig. 4B) for a reason not understood.

Energy Requirement for the C_iCycling Activity-- To distinguish between ATP hydrolysis and µH⁺ as the direct source of energy for CO₂ uptake, we have examined the effects of drugs that specifically inhibit ATP synthesis as well as uncouplers that dissipate the µH⁺. Arsenate and DCCD inhibit the formation of ATP at the substrate level and proton gradient driven synthesis respectively while hardly affecting µH⁺ (35, 36). The uncouplers CCCP and ammonia, on the other hand, abolish the generation of µH⁺.

Photosynthetic O₂ evolution gradually ceased following addition of arsenate to a cell suspension of Synechococcus PCC7942 (Fig. 5A). The slowness of the response probably reflects the rate of arsenate uptake. Although the ambient [CO_2(dis)] rose after about 100 s, it was still below the CO₂/HCO equilibrium value after photosynthesis had halted completely. The further rise in [CO_2(dis)] after addition of CA confirmed the lack of CO₂/HCO equilibrium due to net CO₂ uptake. Addition of DCCD brought about a rapid rise in [CO_2(dis)] (Fig. 5B), but as in the case of arsenate treatment, [CO_2(dis)] was still well below the equilibrium value after O₂ evolution (CO₂ fixation) had ceased. Following the DCCD treatment, the rate of CO₂ uptake, calculated from the plateaus in the curve before and after the addition of DCCD, declined by 54% (from 240 to 112 µmol CO₂ absorbed mg¹ Chl h¹). At the same time, net O₂ evolution (235 µmol O₂ evolved mg¹ Chl h¹) was replaced by respiratory O₂ uptake (67 µmol O₂ absorbed mg¹ Chl h¹). Supply of MV led to a slight rise in [CO_2(dis)] but the rate of increase was much lower than that observed in the absence of DCCD (Fig. 5B) possibly because of lack of sufficient ATP to drive HCO uptake (and consequently CO₂ extrusion). Subsequent CA supply sharply increased the [CO_2(dis)] again indicating that the latter was below the equilibrium value due to net CO₂ uptake. On the addition of the proton conductor CCCP, the rate of O₂ evolution rose transiently (Fig. 5C), most probably because of stimulation of electron transport due to dissipation of the trans-thylakoid µH⁺. Photosynthetic O₂ evolution then ceased, but O₂ uptake at a rate similar to that of dark respiration was detectable. The [CO_2(dis)] rose almost immediately upon addition of CCCP, briefly overshooting equilibrium value. The [CO_2(dis)] level trace resembles that observed when photosynthetic ET is halted by darkening (see Fig. 1). Similar results were obtained using ammonium chloride (not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   A, the effect of arsenate (As), B, N,N,-dicyclohexylcarbo-diimide (DCCD), and C, carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the displacement of external [CO2(dis)] from chemical equilibrium and on O2 evolution by Synechococcus PCC 7942. On addition of CA external [CO2(dis)] returned to equilibrium. Cell density corresponded to 10 µg Chl/ml. Light intensity (in µmol photons m2 s1) is provided at the top.

The [CO_2(dis)] was below CO₂/HCO equilibrium even when the internal ATP was largely exhausted as indicated by the cessation of O₂ evolution following the arsenate or DCCD treatments (Fig. 5, A and B). On the other hand, the uncouplers abolished both CO₂ uptake and fixation (Fig. 5C). These data suggest that CO₂ uptake depends on a µH⁺ and that direct involvement of ATP hydrolysis in the displacement of [CO_2(dis)] below equilibrium is therefore unlikely.

	DISCUSSION

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Severe inhibition of CO₂ uptake by the aquaporin blocker (Fig. 3) suggests that these channels form a major route for CO₂ entry to high-CO₂-grown Synechococcus cells. Because passage of CO₂ through aquaporins is presumably passive either by diffusion or by mass flow together with water molecules, CO₂ transport mediated by specific membrane entities would appear to play a minor role, if any, unless the aquaporin blocker also specifically inhibits these entities.

Results presented in this work confirm that net CO₂ uptake by Cyanobacteria is not directly linked to CO₂ fixation and may proceed in its absence (Figs. 1, 2, and 4). Under low, but not high light intensity, CO₂ uptake by mutant IL-3, which had been maintained at CO₂ concentration lower than its threshold for CO₂ fixation, was faster than in the wild type (Fig. 2). This may reflect consumption of NADPH and/or dissipation of µH⁺ to support CO₂ fixation in the wild type. As discussed below, CO₂ uptake may well be driven by electron transport-dependent µH⁺ (6). At saturating light intensity, net CO₂ uptake was similar in the wild type and in mutant IL-3 suggesting common limitation by the rate of conversion of CO₂ to HCO.

In view of the queries recently raised as to the role of PSI as the major energy source for CO₂ uptake (24), we summarize the evidence in favor of this role obtained in the present investigation as follows. 1) CO₂/HCO disequilibrium consequent on net CO₂ uptake was formed even in the presence of DCMU when reduced duroquinone (Fig. 4A) or dithiothreitol (not shown) was added. 2) C_i cycling was absent in the psaA/B mutant, lacking PSI activity even in the presence of DMBQ, which enabled a high flow of electrons via PSII (Fig. 4B); 3) Synechocystis PCC6803 mutant M55 or the Synechococcus PCC7942 mutant N5 in which ndhB had been inactivated (37, 38) are defective in cyclic PSI ET. Although they exhibit photosynthetic carbon fixation when supplied with elevated CO₂ levels (38), they are unable to displace the CO₂/HCO equilibrium. 4) Draining electrons from PSI by means of artificial acceptors switched Synechococcus PCC7942 from net CO₂ uptake to net HCO uptake (Fig. 4, C and D, see also Ref. 23).

These observations together with those reported elsewhere (23, 25, 39) provide a very strong case for the central role of PSI ET in driving CO₂ uptake. The question is, therefore, how these observations can be reconciled with the contrasting effects of inactivation of NDH-1 components on CO₂ uptake and on P700 oxidation in Synechocystis PCC6803 (24). Another problem is that the results presented here indicate that a µH⁺ generated by photosynthetic ET serves as the direct source of energy for CO₂ uptake (Fig. 5). This finding is consistent with the predictions of our model (6), but the exclusive dependence of CO₂ uptake on PSI ET has to be reconciled with the accepted notion that both linear and cyclic PSI ET lead to the formation of a µH⁺ across the thylakoid membrane. Both problems are addressed in the following discussion.

To account for the effect of mutation in a component of NDH-1 on CO₂ uptake and on P700 oxidation, Klughammer et al., (26) suggested that in Synechococcus PCC7002 one type of NDH-1 essential for cyclic ET is located on the thylakoid, whereas another type engaged in CO₂ uptake is located on the cytoplasmic membrane. However, immunolocalization studies have demonstrated that a component of NDH-1, NdhB, critical for CO₂ uptake is exclusively located in the thylakoid membrane (25).

The possibility should be considered that only a small fraction of the PSI population is engaged in CO₂ uptake. Depression of CO₂ uptake in the ndhD3/ndhD4 mutant (24) was in fact associated with some decline in cyclic PSI activity, possibly reflecting this small fraction of PSI. In the ndhD1/ndhD2 mutant where cyclic PSI activity was largely depressed, CO₂ uptake was little affected presumably because the PSI units engaged in CO₂ uptake were still operating. The marked dependence of the PSI/PSII ratio on the growth conditions, particularly CO₂ concentration (22) and salinity (40), would be in accordance with such a suggestion. The observation that the distribution of PSI in Synechococcus PCC7942 is heterogeneous, indicated by the higher abundance of PSI in peripheral as compared with inner thylakoid membranes (41), also lends support to the notion that the PSI population may be functionally heterogeneous. This heterogeneity might relate to different routes for electron flow from NADPH to plastoquinone (42, 43).

Different routes for electron flow might also be the basis of the differences in CO₂ uptake between the various ndhD mutants (24). Uptake of CO₂ was observed in each of the single mutants ndhD3 and ndhD4 but not in the double mutant (24). Ogawa et al.³ concluded that two discrete systems for CO₂ uptake operate in Synechocystis PCC6803. This is based on the differing kinetic parameters for CO₂ uptake between mutants ndhD4 and ndhD3 and the inducibility of CO₂ uptake by low CO₂ conditions in the wild type and in mutant ndhD4 but not in mutant ndhD3. The NdhD3- and NdhD4-dependent CO₂ uptake systems may constitute alternative PSI-dependent routes for electron flow. Quantitative consideration shows that the residual rate of P700 oxidation (i.e. the PSI cyclic electron flux) in the ndhD1/ndhD2 (24) was too low to account for the rate of CO₂ uptake in this mutant. This may indicate the presence of an alternative acceptor of electrons from the NdhD3- and NdhD4-dependent CO₂ uptake systems such as succinate:quinol oxidoreductases (44), a possibility currently being examined.

It has recently been proposed (6) that CO₂ is converted to HCO in alkaline domains on the stromal face of the thylakoid membrane (see the Introduction). Conversion of CO₂ to HCO in such domains maintains the inward diffusion gradient for CO₂ and a cytoplasmic CO₂ concentration below that of equilibrium with HCO. Withdrawal of electrons from plastoquinone by either DMBQ or MV (Fig. 4) would prevent the formation of these alkaline domains and thus also of CO₂ uptake. The exclusive reliance of CO₂ uptake on PSI-generated µH⁺ probably involves an electron carrier yet to be identified. In view of the finding that NdhD3 and NdhD4 are essential components of two CO₂ uptake systems (Ogawa et al., submitted) but not of the respiratory electron path they are likely candidates for the formation of the alkaline domains during PSI ET. Another candidate is ferredoxin-NADPH reductase. Recent studies (43) demonstrated that linear ET was functional but that the plastoquinone-cytochrome b6f complex reductase step of cyclic PSI was defective in a mutant where the N-terminal of ferredoxin-NADPH reductase was truncated preventing its association with the thylakoids).

	ACKNOWLEDGEMENTS

We thank Dr. W. Vermaas who kindly provided us with mutant psaA/B of Synechocystis PCC 6803. The water channel blocker, p-chloromercuriphenylsulfonic acid, was generously provided by Prof. N. Murata, Okazaki, Japan.

	FOOTNOTES

^* This research was supported by grants from: the United States-Israel Binational Science Foundation (BSF); Program MARS2, a cooperation between the German Bundes Ministerium fur Bildung Wissenschaft, Forschung und Technologie (BMBF) and the Israeli Ministry of Science (MOS); and by the Ministry of Science and Culture of the State of Niedersachsen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Plant Sciences, The Hebrew Univ. of Jerusalem, 91904 Jerusalem, Israel. Tel.: 972-2-6585234; Fax: 972-2-6584463; E-mail: aaronka@vms.huji.ac.il.

Published, JBC Papers in Press, April 10, 2001, DOI 10.1074/jbc.M101973200

² D. Tchernov, Y. Helman, B. Luz, I. Ohad, L. Reinhold, and A. Kaplan, manuscript in preparation.

³ M. Shibata, H. Ohkawa, T. Kaneko, H. Fukuzawa, S. Tabata, A. Kaplan, and T. Ogawa, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: CA, carbonic anhydrase; WCB, water channel blocker; PSI, photosystem I; PSII, photosystem II; ET, electron transport; chl, chlorophyll; IAC, iodoacetamide; DCMU, 3- (3,4-dichlorophenyl)-1,1-dimethylurea; DMBQ, 2,5-dimethyl-p-benzoquinone; MV, methyl viologen; CCCP, carbonyl cyanide m-chlorophenylhydrazone.

	REFERENCES

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