BioSystems 103 (2011) 224–229
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BioSystems
journal homepage: www.elsevier.com/locate/biosystems
Optimization of CO2 fixation in photosynthetic cells via thermodynamic buffering
Abir U. Igamberdiev a,∗ , Leszek A. Kleczkowski b
a
b
Department of Biology, Memorial University of Newfoundland, Main Campus, St. John’s, NL A1B 3X9, Canada
Department of Plant Physiology, Umeå Plant Science Centre, University of Umeå, 901 87 Umeå, Sweden
a r t i c l e
i n f o
Article history:
Received 14 June 2010
Accepted 1 October 2010
Keywords:
Adenylate kinase
Carbonic anhydrase
Homeostatic flux control
Photorespiration
Thermodynamic buffering
a b s t r a c t
Stable operation of photosynthesis is based on the establishment of local equilibria of metabolites in
the Calvin cycle. This concerns especially equilibration of stromal contents of adenylates and pyridine
nucleotides and buffering of CO2 concentration to prevent its depletion at the sites of Rubisco. Thermodynamic buffering that controls the homeostatic flux in the Calvin cycle is achieved by equilibrium
enzymes such as glyceraldehyde phosphate dehydrogenase, transaldolase and transketolase. Their role is
to prevent depletion of ribulose-1,5-bisphosphate, even at high [CO2 ], and to maintain conditions where
the only control is exerted by the CO2 supply. Buffering of adenylates is achieved mainly by chloroplastic adenylate kinase, whereas NADPH level is maintained by mechanisms involving alternative sinks for
electrons both within the chloroplast (cyclic phosphorylation, chlororespiration, etc.) and shuttling of
reductants outside chloroplast (malate valve). This results in optimization of carbon fixation in chloroplasts, illustrating the principle that the energy of light is used to support stable non-equilibrium which
drives all living processes in plants.
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Although living systems operate far from thermodynamic equilibrium, non-equilibrium fluxes that support their metabolism
should be steady (Igamberdiev and Kleczkowski, 2009). The external energy is used primarily to support stable non-equilibrium state
(Bauer, 1982), which becomes an internal source of work conducted
by a biosystem. This can be reached, in particular, via a “fitting
function” of special thermodynamic buffering enzymes that equilibrate fluxes of load and fluxes of consumption of major metabolic
components, e.g., ATP and pyridine nucleotides (Igamberdiev and
Kleczkowski, 2003). Thermodynamic buffering generates steady
metabolic fluxes via local equilibrations and regulated uncoupling or “slippage” of the oxidation processes from ATP synthesis
(Igamberdiev and Kleczkowski, 2009). Understanding mechanisms
of such equilibration will help in developing a computational
approach for calculating major parameters of metabolism. This
may include metabolic flux analyses which provide tools to measure and model metabolism and its functions (Allen et al., 2009).
These approaches consider a co-existence of both equilibrium
and non-equilibrium reactions in complex metabolic networks
(Galimov, 2004). Equilibration of metabolites by the equilibrium
Abbreviations: AK, adenylate kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PFK, phosphofructokinase; PGA, 3-phosphoglycerate; RuBP, ribulose
bisphosphate; Rubisco, RuBP carboxylase/oxygenase.
∗ Corresponding author. Tel.: +1 709 864 4567; fax: +1 709 864 3018.
E-mail address: [email protected] (A.U. Igamberdiev).
0303-2647/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.biosystems.2010.10.001
(thermodynamic buffer) enzymes (Stucki, 1980) leads to a steady
non-equilibrium flux through irreversible reactions and prevents
depletion of substrates for non-equilibrium enzymatic reactions.
This general idea of a homeostatic flux control (Fridlyand and
Scheibe, 2000) provides a metabolic substantiation for the concept
of homeorhesis (Waddington, 1968), i.e. of temporal steady trajectories in the development of biological systems. The key properties
of biochemical networks should be robust in order to ensure their
proper functioning (Barkai and Leibler, 1997) and to maintain stable
non-equilibrium state (Bauer, 1982).
Photosynthetic metabolism is an example of optimized homeostatic flux where carbon is efficiently fixed using the energy (ATP)
and the reducing power (NADPH) formed in light reactions. For
optimization of the flux, the following conditions should be satisfied: (1) the homeostatic flux control of metabolic pathways,
including the Calvin cycle and photorespiration; (2) the buffering
of adenylates both within their own pool and in relation to the pool
of pyridine nucleotides, thus providing the maintenance of a stable
ATP/NADPH ratio; (3) the buffering of reductants including pyridine nucleotides and ascorbate; (4) the buffering of the substrate
of photosynthesis (CO2 ). In this paper, we discuss possible mechanisms providing the thermodynamic buffering and maintaining
stable non-equilibrium during photosynthesis.
2. Homeostatic Flux Control in the Calvin Cycle
Metabolic cycles are organized in a way that they maintain
stable non-equilibrium flux by providing a steady turnover of
substrates through the cycle (Igamberdiev, 1999; Qian et al.,
A.U. Igamberdiev, L.A. Kleczkowski / BioSystems 103 (2011) 224–229
2003). Every metabolic cycle contains both equilibrium and nonequilibrium reactions. For instance, in the citric acid cycle, the
equilibrium reactions are catalyzed by malate dehydrogenase,
fumarase, aconitase (MacDougall and ApRees, 1991; Hagedorn
et al., 2004) and NADP-isocitrate dehydrogenase (Igamberdiev
and Gardeström, 2003). As earlier suggested (Igamberdiev, 1999),
the simplest metabolic substrate cycle includes one irreversible
(non-equilibrium) and one reversible (equilibrium) reactions.
Examples include the cycle composed of irreversible NAD- and
reversible NADP-isocitrate dehydrogenase reactions (Igamberdiev
and Gardeström, 2003), the glycolate oxidase/NAD(P)H-glyoxylate
reductase cycle (Kleczkowski and Givan, 1988; Kleczkowski and
Randall, 1988; Igamberdiev and Lea, 2002), and ATP- and PPidependent phosphofructokinase (PFK) cycle (Huang et al., 2008).
Most metabolic cycles/pathways evolved via transformations of
these simple substrate cycles (Igamberdiev, 1999).
The commonly accepted idea that irreversible reactions in
cycles limit their turnover has been shown to be generally incorrect (Fridlyand and Scheibe, 1999a, 2000; Fridlyand et al., 1999;
Morandini, 2009). There are strong theoretical arguments against
the idea that highly regulated enzymes catalyzing reactions far
from equilibrium must be considered a priori rate limiting. Conversely, contrary to accepted wisdom, the reactions close to
equilibrium frequently limit flux when the amount of the enzyme
is reduced (Morandini, 2009). Changes in provision of a substrate
to the cycle or pathway are controlled by thermodynamic buffering reactions that can reverse a product back to the substrate. Also,
when levels of a metabolite provided by the equilibrium enzyme
are insufficient for maximum flux, the cycle flux could still be
increased by involving separate depots of stored intermediates. For
the Krebs cycle, this is achieved by the involvement of large pools
of malate, citrate and other stored acids (including fumarate, transaconitate, isocitrate in some species). For the Calvin cycle, the pool
of triose phosphates (e.g. from the cytosol), and probably ribose5-phosphate and erythrose-4-phosphate, may play a similar role.
Another example is photorespiration which eventually supplies 3phosphoglycerate (PGA) back to the Calvin cycle (Kleczkowski and
Givan, 1988; Igamberdiev and Lea, 2002).
Fridlyand and Scheibe (1999a, 2000) considered the importance
of turnover times (or pool sizes) of the Calvin cycle intermediates
to optimize metabolism. Presumably, there are mechanisms for
adjusting uniformly the rates of individual reactions inside the cycle
(Fridlyand and Scheibe, 1999a, 2000). Indeed, the carboxylation
rate can completely limit the rate of photosynthesis, at least at low
[CO2 ] when the rate of RuBP carboxylation is strongly limited by
Rubisco (Farquhar et al., 1980; Woodrow and Berry, 1988). Even at
high [CO2 ], it has been proposed that RuBP is not limiting, but rather
it serves as a gatekeeper and, thus, the paramount controller of
CO2 fixation under any condition (Farazdaghi and Edwards, 1988;
Farazdaghi, 2009). Such a model substantiates the robustness of
the Calvin cycle and its relative insensitivity to precise values of
biochemical parameters.
Steady operation of the Calvin cycle assumes that the contribution of every individual step to the turnover period ( i /) is
determined by the relative concentration of the metabolite at a
given step. In these conditions, i / = Si /, where Si is the concentration of the metabolite at a given step, i is the transient time
for this step, is the sum of the lifetimes of all metabolite pools
(it can be regarded as the turnover period of the cycle or the mean
time of the turnover of one acceptor molecule in the cycle), and is the total concentration of metabolites in the cycle (Fridlyand and
Scheibe, 1999a).
Main equilibria in the Calvin cycle are achieved by glyceraldehyde dehydrogenase (GAPDH), transketolase, and aldolase. GAPDH
catalyses the reversible reduction of 1,3-bisphosphoglycerate. Its
rate depends on the ATP/ADP ratio (really MgATP/MgADP) and
225
NADPH NADP+
ATP
ADP
PGA
P2GA
GAP
Sugar-P2
ADP
CO2
NADH NAD+
RuBP
Pi
ATP
Sugar-P
ADP
ATP
Photosynthec products
Fig. 1. A generalized structure of the Calvin cycle. The bypasses forming substrate
cycles and catalyzed by chloroplastic glycolytic enzymes are shown by dotted lines.
Abbreviations: RuBP – ribulose-1,5-bisphosphate, PGA – 3-phosphoglycerate, P2 GA
– 1,3-bisphosphoglycerate, GAP – glyceraldehyde-3-phosphate.
levels of 1,3-bisphosphoglycerate, but considering an unlimited
formation of the latter in the kinase reaction, it depends on
PGA formed in the Rubisco reaction. Phosphoglycerate kinase and
triose-phosphate isomerase exhibit very high activities and do
not exert any limitation on the Calvin cycle turnover (Fridlyand,
1992). The equilibrium transketolase and transaldolase reactions,
together with phosphopentose isomerase result in the production
of ribulose-5-P which, after entering the irreversible phosphoribulokinase reaction, forms RuBP, the substrate for Rubisco.
The net result of the coupling of equilibrium and nonequilibrium reactions in the Calvin cycle is to produce a sufficient
amount of RuBP for Rubisco to adjust rates of CO2 assimilation to
turnover rates of the cycle intermediates (Fridlyand and Scheibe,
1999a). In conditions of homeostatic operation of the Calvin cycle,
Rubisco becomes its main controlling point through CO2 concentration (and also the O2 level). This would not be possible in case of
a direct reduction of CO2 via formate and formaldehyde, the mechanism largely abandoned by living matter, although there are some
indications that it may have a minor contribution to photosynthetic
metabolism (reviewed in Igamberdiev et al., 1999).
From the analysis of operation of the Calvin cycle (Fig. 1), we
see the basic principle of the structure of the metabolic cycle. Any
cycle consists of both equilibrium and non-equilibrium reactions.
The equilibrium enzymes in cycles buffer them, while the nonequilibrium enzymes drive them. The equilibrium reactions are
generally limiting because they create equilibrium concentrations
to supply substrates for fast non-equilibrium fluxes. These equilibria set turnover times for cycles and provide conditions when the
cycle operation is limited only by the availability of the entry substrate (in the Calvin cycle – CO2 ) and supply of the energy (ATP)
and the reductant (NADPH) at optimum rate and stoichiometry.
This can be achieved via mechanisms that buffer supply of ATP,
NADPH, and CO2 . We discuss them below.
3. Buffering of Adenylates in the Chloroplast by Adenylate
Kinase
To provide the stable and sustainable rate of CO2 fixation,
it is important that the ATP/ADP ratio in chloroplasts is maintained at certain optimal level. Too low level will result in the
suppression of Calvin cycle turnover at the levels of PGA phosphorylation/reduction and ribulose-5-P phosphorylation (Fridlyand and
Scheibe, 1999a), while too high level will result in the ADP depletion for ATP synthesis and in the depletion of Mg2+ in the stroma
(Igamberdiev and Kleczkowski, 2006). Under non-photorespiratory
conditions, the values of ATP/ADP in leaf chloroplasts are relatively
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A.U. Igamberdiev, L.A. Kleczkowski / BioSystems 103 (2011) 224–229
Operation of ETC in chloroplast
Oxygen reduction
(Mehler reaction)
Nitrite reduction
Malate valve
Generation of membrane potential and ∆ pH
ATP synthesis by ATP synthase
e- from H2O
Fdr
NADPH
Cyclic flow
Chlororespiration
PGA reduction
Equilibrium of adenylates by AK
Establishment of stromal ATP/ADP ratios
Establishment of stromal [Mg2+]
Regulation of Rubisco
and other Calvin cycle enzymes
Sustainable CO2 fixation
in the Calvin cycle
Fig. 2. Adenylate buffering in chloroplasts. Schematic representation of the links
between AK equilibrium, chloroplast ATP/ADP ratio, concentration of free magnesium, and control over Rubisco activity.
low (on the order of 1.1–1.3), but they increase during photorespiration (up to 3–4) (Gardeström and Wigge, 1988; Igamberdiev
et al., 2001). The main mechanism to balance the ATP/ADP ratio
upon changes of metabolite concentrations or upon limitations of
the electron transport is the thermodynamic buffering of adenylates by adenylate kinase (AK) (Igamberdiev and Kleczkowski, 2003,
2006) (Fig. 2). AK is a strong regulator of concentrations of adenylates and Mg2+ in the cells, in particular in chloroplast stroma
and the mitochondrial intermembrane space. During the transition from darkness to light, when a significant increase of [ATP]
occurs, there could be a delay in photosynthesis (photosynthetic
induction) caused by a decrease in stromal [Mg2+ ], with both the
adenylate composition and [Mg2+ ] controlled by AK equilibrium
(Igamberdiev and Kleczkowski, 2001; Igamberdiev et al., 2001).
This delays the Mg2+ -dependent activation of Rubisco and other
enzymes that require Mg2+ for activity.
Quantitative analyses by Fridlyand (1992) and Fridlyand et al.
(1997) have demonstrated that, at the level of PGA reduction, the
rate of GAPDH reaction (v) completely depends both on [3-PGA] and
the ATP/ADP ratio. Thus, v = KVmax [PGA]([ATP]/[ADP]), where K is
the apparent constant for PGA reduction, and Vmax is the activity of
GAPDH. More correctly, the MgATP/MgADP ratio drives this reaction (Igamberdiev and Kleczkowski, 2001). To keep the steady flow
through the Calvin cycle, this ratio should be maintained steady.
Under photorespiratory conditions, the flux of carbon turns to the
glycolate pathway and [ATP] rises, thus also decreasing the Calvin
cycle capacity (lower [Mg2+ ]). This relatively lower flux through the
Calvin cycle is accompanied by the supply of ATP for refixation of
photorespiratory ammonia. The calculated values of [Mg2+ ] in low
[CO2 ] are lower than in high [CO2 ] (Igamberdiev and Kleczkowski,
2001), thus limiting the operation of Rubisco and the Calvin cycle.
An important factor that affects regulation by the ATP/ADP ratio
is the direct transport of ATP from the chloroplast (Flügge and
Heldt, 1984). The capacity of chloroplastic adenylate transporters
is usually limited under light conditions, and the energy (e.g. for
sucrose synthesis) is largely transported as a triose-P in exchange
with phosphate. This means that the transport through chloroplast
membrane cannot exert a significant control over intrachloroplastic ATP and ADP levels. This, in turn, substantiates the view that
the supply of ATP to the cytosol may depend to a large extent on
mitochondrial ATP synthesis (Gardeström and Wigge, 1988). This
also means that the buffering of adenylates for steady fluxes of
the photosynthetic phosphorylation and the Calvin cycle occurs
Fig. 3. A generalized scheme of electron sinks in chloroplasts.
via the chloroplastic AK system, while the regulation of the reductant pool takes place both inside and outside the chloroplasts (the
latter through the malate valve) (Krömer and Scheibe, 1996). In
C4 plants, AK has an additional task of recycling AMP produced by
pyruvate-phosphate-dikinase reaction and is abundant particularly
in mesophyll chloroplasts (Kleczkowski and Randall, 1986; Wild et
al., 1997).
4. Regulation of the ATP/NADPH Ratio in Photosynthetic
Cells
Stable operation of the Calvin cycle is supported by stable
ATP/NADPH ratio in chloroplasts (Noctor and Foyer, 1998, 2000).
Assimilation of one CO2 molecule requires three ATP and two
NADPH, while photorespiration demands additional ATP molecules
for glycerate phosphorylation and for refixation of photorespiratory ammonia. Nitrate and nitrite reduction provide additional
sinks for the reductant during photosynthesis (Noctor and Foyer,
1998).
Stromal ATP/NADPH ratio is adjusted by various mechanisms
which direct electrons to acceptors other than CO2 . The adjustment can be achieved by cyclic electron flow around PSI (mediated
by ferredoxin: NADPH reductase, NAD(P)H dehydrogenase, or
putative ferredoxin: plastoquinone reductase), by the reduction
of O2 in the Mehler reaction, or by the malate valve (Fig. 3).
The cyclic electron flow generates ATP without reducing NADP+ ,
while the Mehler reaction provides the sink for electrons, linking it to another redox buffer – ascorbate (Ort and Baker, 2002).
Chlororespiration (mediated by NAD(P)H dehydrogenase) oxidizes
NAD(P)H in the chloroplast without ATP synthesis, while the malate
valve represents a mechanism transporting the reductant outside
the chloroplast. It can be used for hydroxypyruvate reduction in
peroxisomes (Hanning and Heldt, 1993) or for oxidation in the
electron transport chain in mitochondria, with the latter process coupled or non-coupled to ATP synthesis (Igamberdiev et al.,
1998). Cyclic phosphorylation increases [ATP] at constant [NADPH],
while chlororespiration and other non-coupled pathways decrease
[NADPH] (reduction level) without [ATP] increase. The antisense
reduction of NADPH-glyceraldehyde phosphate dehydrogenase,
which results in slowing down the Calvin cycle and a lower rate
of NADPH consumption, leads to an increase in the rate of cyclic
phosphorylation around PSI (Livingston et al., 2010). This may be
directly related to the adjustment of the ATP/NADPH ratio in these
plants. Reduction of O2 through the Mehler reaction results in the
decrease of NADP reduction level.
Environmental stresses such as photoinhibition, high temperatures, drought, or high salinity tend to stimulate the activity
of alternative PSI-driven electron transport pathways. Thus, the
energetic and regulatory functions of these pathways must be an
integral part of photosynthetic organisms, providing additional
flexibility to environmental stress (Bukhov and Carpentier, 2004).
Unlike ferredoxin-dependent cyclic electron transport, the pathways supported by NAD(P)H oxidation can function in the dark and
are likely involved in chlororespiratory-dependent energization of
A.U. Igamberdiev, L.A. Kleczkowski / BioSystems 103 (2011) 224–229
the thylakoid membrane supporting carotenoid biosynthesis and
maintaining thylakoid ATPase in active state. The rate of NAD(P)Hdependent pathways under light depends largely on NAD(P)H
accumulation in the stroma (Bukhov and Carpentier, 2004).
The transfer capacity of the malate valve is estimated not to
exceed 20 ␮mol (mg Chl)−1 h−1 (or 5% of the electron transport)
under normal physiological conditions (Fridlyand et al., 1998).
The valve is a most flexible system of transport of redox equivalents. It can either increase redox level in another compartment, or
lead to extra ATP synthesis in mitochondria, serving either as the
transporter of redox equivalent or energy equivalent. By accepting reducing equivalents, malate valve also supports ATP synthesis
within the chloroplast.
Fridlyand and Scheibe (1999b) developed the equations for
electron transport to ferredoxin, nitrite reduction, cyclic electron transport, malate valve, PGA reduction, and O2 reduction
in chloroplast. They calculated control coefficients for different
electron fluxes and showed that chloroplasts can maintain the
NADPH/NADP+ ratio constant within a wide range of light intensities. In their model, the irreversible exergonic reactions that are
not coupled to ATP synthesis can be tightly regulated by shifts in
the balance between the reactions of load and consumption. This so
called “regulated uncoupling” occurs, in general, if one of two coupled reactions of a cyclic process proceeds without its counterpart.
The switch between the cyclic and the non-cyclic phosphorylation is strongly regulated via special protein factors (DalCorso et
al., 2008). The data of Laisk et al. (2007) show that the alternative and cyclic electron flow necessary to compensate variations
in the ATP/NADPH ratio represent only a few percent of the linear
flow of energy-dissipating processes around PSI and PSII at light
saturation.
The modulation of proton influx through cyclic electron flow
around PSI is suggested to play a role in regulating the ATP/NADPH
output ratio of the light reactions (Avenson et al., 2005; Cruz et
al., 2005). In dark-adapted leaves, the cyclic flow operates at maximum rate, owing to the partial inactivation of the Calvin cycle. For
increasing time of illumination, the activation of the Calvin cycle,
and thus that of the linear flow, is associated with a subsequent
decrease in the rate of the cyclic flow. Under steady-state conditions of illumination, the contribution of cyclic flow to PSI turnover
increases as a function of the light intensity (from 0 to approximately 50% for weak to saturating light, respectively). Lack of CO2
is associated with an increase in the efficiency of the cyclic flow.
ATP concentration could be one of the parameters that control the
transition between the linear and the cyclic flow modes (Joliot and
Joliot, 2006). The cyclic electron flow can operate under aerobic
conditions and support a simple competition model, where the
excess reducing power is recycled to match the demand for ATP
(Alric et al., 2010). Chlororespiration likely plays a role in the regulation of photosynthesis by modulating the activity of cyclic electron
flow around PSI (Peltier and Cournac, 2002).
It is important to mention here that the buffering of adenylates
and pyridine nucleotides may take place within the Calvin cycle
through the corresponding substrate cycles. For this, the enzymes
catalyzing reverse reactions are important. These are mainly glycolytic enzymes catalyzing the reactions that are opposite in the
direction when compared to corresponding reactions of the Calvin
cycle. Trimming and Emes (1993) detected all of the glycolytic
enzymes, except hexokinase and phosphoglyceromutase, in pea
plastids. Garland and Dennis (1980) compared the properties of
plastidic and cytosolic PFKs and found kinetic and regulatory differences between the two isoenzymes: the plastid PFK was sensitive
to inhibition by ATP and was phosphate-activated, in contrast to
the cytosolic isoenzyme. PFK may convert not only fructose-6-P
to fructose-1,6-bisP but also sedoheptulose-7-P to sedoheptulose1,7-bisP (Karadsheh et al., 1973). The latter reaction is carried out
227
in the opposite direction than that of the sedoheptulose bisphosphatase in the Calvin cycle. This means that [ATP] can be finely
controlled within the stroma. The NAD-dependent GAPDH present
in the plastids, in conjunction with the corresponding NADPHdependent enzymes of the Calvin cycle, can balance redox levels
of pyridine nucleotides (NAD and NADP) and also ATP/ADP ratios.
These glycolytic reactions bypassing the reactions of the Calvin
cycle are shown by dot lines in Fig. 1. They can contribute to finetuning of the metabolic flux through the cycle.
5. CO2 Buffering as a Condition for Stable Operation of the
Calvin Cycle
Rubisco, the first enzyme of the Calvin cycle, uses RuBP as an
acceptor for either carboxylation (with CO2 ) or oxygenation (with
O2 ) reactions. The magnitude of each reaction depends on the
[CO2 ]/[O2 ] ratio in the vicinity of Rubisco which is determined by
the atmospheric concentrations of both gases, their diffusion to
chloroplasts, and the degree of CO2 depletion by Rubisco itself. The
carboxylation and oxygenation reactions are each followed by a
series of metabolic steps referred to as the Calvin cycle (carboxylation) and the glycolate cycle (oxygenation). The latter pathway
involves several compartments and brings about (among other
things) a release of CO2 from mitochondria (photorespiration).
In vivo concentration of Rubisco in chloroplast stroma is up
to three orders of magnitude higher than that of its gaseous substrates and approximately equal to that of RuBP (Jensen and Bahr,
1977; Pickersgill, 1986). In addition, stromal [CO2 ] is below its
Km value with Rubisco (Woodrow and Berry, 1988), implying that
any depletion of CO2 would result in an immediate decrease of
Rubisco activity and, thus, decreasing the rate of photosynthesis.
This, in turn, implies that for stable operation of Rubisco, a feedback mechanism should exist that supplies CO2 internally from the
reactions where it is released (respiration and photorespiration).
In other words, to prevent depletion during the active photosynthesis process and to support stable operation of the Calvin
cycle, the CO2 concentration should be buffered in the vicinity of
Rubisco. In this buffering process, the competition between two
substrates (CO2 and O2 ) and the delay in CO2 release, when Rubisco
takes O2 , results in a kinetic behavior that leads to generation of
oscillations (Roussel et al., 2007; Roussel and Igamberdiev, 2011),
which may be an essential consequence of the Rubisco reaction
mechanism.
For every four carbons entering the glycolate cycle due to oxygenase activity of Rubisco, one CO2 is released (in mitochodria).
This released carbon is not necessarily lost to the atmosphere –
some estimates give values as high as over 80% for the efficiency of
refixation of the photorespiratory carbon (Pinelli and Loreto, 2003).
Carbon released due to the mitochondrial Krebs cycle activity in the
light can also be refixed. For refixation to occur, the CO2 needs to
be released from mitochondria, cross the cytosol, and end up in the
stromal compartment. In the CO2 refixation process, an important
role may be played by carbonic anhydrase isozymes which equilibrate pools of CO2 and bicarbonate (Raven, 2001; Riazunnisa et
al., 2006). In this way, the CO2 released in mitochondria can be
transported as bicarbonate, the process in which mitochondrial,
cytosolic, chloroplast carbonic anhydrases and mitochondrial and
chloroplast bicarbonate transporters work together preventing CO2
loss and facilitating CO2 refixation (Fig. 4). The carbonic anhydrase
reaction may also be involved in a kind of the CO2 concentrating mechanism of C3 plants. In this process, localized entirely in
chloroplasts, the stromal and thylakoid-based carbonic anhydrases
cooperate to pump bicarbonate into thylakoids in exchange for CO2 ,
thus effectively increasing [CO2 ] near Rubisco (Fridlyand and Kaler,
1987; Hanson et al., 2003). The cooperation of Rubisco and carbonic
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A.U. Igamberdiev, L.A. Kleczkowski / BioSystems 103 (2011) 224–229
Fig. 4. The scheme showing CO2 buffering in photosynthetic cell. Rubisco participates both in reactions with CO2 (as carboxylase) and O2 (as oxygenase), the latter
eventually leading to the appearance of glycine in the mitochondria and, subsequently, to photorespiration. The CO2 produced by photorespiration is transported
through the cytoplasm to the chloroplasts either directly or as bicarbonate after
conversion by the mitochondrial carbonic anhydrase. The equilibria between CO2
and bicarbonate established by corresponding carbonic anhydrases are shown in
chloroplasts, mitochondria and cytosol.
anhydrases in CO2 pumping and refixation constitutes the pivotal
mechanism providing the essential CO2 buffering that supports and
maintains the stable operation of the Calvin cycle.
6. Conclusion
The photosynthetic CO2 fixation is controlled by mechanisms
that provide steady homeostatic flux through the Calvin cycle. The
reactions that operate close to the thermodynamic equilibrium,
especially the reduction of PGA and the transketolase reaction can
significantly influence the total turnover period in the Calvin cycle
and provide optimized CO2 fixation rate. In this optimization, the
buffering of adenylates and magnesium (via AK), of NADPH (via
different reactions of slippage and regulated uncoupling), and of
CO2 (via the photorespiratory feedback) work together to make
the CO2 fixation steady during photosynthesis. In this way, the
external energy (light) is used for the maintenance of the stable
non-equilibrium state which, in turn, drives living processes in
plants.
Acknowledgments
This work was supported by the Natural Sciences and Engineering Research Council of Canada (to A. U. I.) and by the Swedish
Research Council (to L. A. K.).
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