Plant Physiology - Musienko M. M. 2001

Photosynthesis: Physiological, Biochemical, and Ecological Aspects
Dark Stage of Photosynthesis. Reductive Pentose Phosphate Cycle

All living organisms possess only a single primary carboxylation mechanism. It is primary in the sense that it gives rise to everything else. This is the pathway for The Biosynthesis of all carbon-containing Organic compounds from CO₂, known as The Calvin Cycle or the reductive pentose phosphate cycle (RPP cycle). The universality of this cycle indicates that it emerged in organisms significantly more primitive than eukaryotic Algae and plants. Photosynthetic Bacteria (with the exception of cyanobacteria) that perform photoreduction are capable of fixing and reducing CO₂ in certain cases.

The soluble Cofactors NADPH and ATP (products of the light-dependent stage) serve as a source of reducing equivalents and energy for The conversion of CO₂ into sugar phosphates within the METABOLISM/14.html">Chloroplasts. The Enzymes catalyzing the individual Reactions of the RPP cycle are Water-soluble and localized in the chloroplast stroma.

There are no photochemical reactions within the Calvin cycle itself, yet the light stages can indirectly influence it. For instance, this occurs through light-induced Changes in the concentration of hydrogen ions and magnesium, as well as the redox state of cofactors such as ferredoxin and NADP. The sequence of reactions was deciphered in the USA by M. Calvin and his coworkers between 1946–1956 (Fig. 70). This work was awarded the Nobel Prize in 1961.

The Mechanism of CO₂ assimilation was deciphered through The Use of carbon-14-labeled (¹⁴C) carbon dioxide, along with chromatographic and autoradiographic analysis Methods. The green unicellular alga Chlorella was used as the research object. Following a brief exposure to light in the presence of ¹⁴CO₂, the plants were fixed with hot alcohol. The alcohol extract was concentrated and then separated on chromatograms. It was discovered that after just 5 seconds of exposure to a ¹⁴CO₂ atmosphere, the majority of the radioactive carbon was concentrated in a three-carbon compound: 3-phosphoglyceric acid (3-PGA). Therefore, it was hypothesized that chloroplasts must contain a specific acceptor compound which, upon interacting with ¹⁴CO₂, forms phosphoglyceric acid (acceptor + ¹⁴CO₂ = PGA). It was observed that after turning off the light, the PGA content continued to rise for some time. Simultaneously, a fairly rapid disappearance of the five-carbon compound, ribulose bisphosphate, was noted. However, within 30 seconds ribulose bisphosphate had vanished, whereas The amount of PGA remained unchanged at that moment. At the same time, in the light, ribulose bisphosphate did not disappear, and its content remained constant. A completely different picture was observed in the absence of carbon dioxide: under those conditions, neither in the dark nor in the light did the ribulose bisphosphate content change. This implies that in the presence of carbon dioxide, ribulose bisphosphate was utilized in the dark to form PGA. Subsequent transformation of PGA requires the presence of light. Analyzing the obtained data, M. Calvin proposed the following sequence of events for the Dark Phase of Photosynthesis.

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Fig. 70. The Calvin-Benson-Bassham cycle (RPP cycle). The number of lines in the arrows indicates how many times the reaction repeats during one full turn of the cycle, in which three molecules of CO₂ are converted into one molecule of GAP

The First stage involves the carboxylation of the primary carbon dioxide acceptor, ribulose-1,5-bisphosphate (RuBP). This attachment reaction is catalyzed by the enzyme ribulose-1,5-bisphosphate carboxylase-oxygenase (RUBISCO), the most abundant protein in the biosphere, which is activated by magnesium. Its Michaelis constant for CO₂ in vitro is 450 µmol. This corresponds to a partial pressure of 1–2% CO₂ at pH 7.5. At a CO₂ concentration of 0.03% (its actual level in atmospheric air), the carboxylation enzyme turns out to be under-saturated. It remains unclear to what extent this occurs in leaf Tissues in vivo. Another quite essential feature of this enzyme is the strong dependence of its mode of action on O₂ (we will examine this dependence in the chapter "Photorespiration").

Initially, an unstable six-carbon molecule (3-carboxy-3-keto-arabinitol-1,5-bisphosphate) is formed, which rapidly decomposes via Hydrolysis into two trioses: 2 molecules of phosphoglyceric acid (PGA):

Next, PGA is reduced to glyceraldehyde 3-phosphate (GAP) using the assimilation energy from the light stage. This occurs in two steps: first, through the involvement of ATP and phosphoglycerate kinase, additional phosphorylation takes place, yielding 1,3-bisphosphoglycerate (1,3-BPG):

In the second stage, 1,3-BPG is reduced to 3-phosphoglyceraldehyde (3-GAP) with the participation of NADPH+H⁺. The reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase, and this is the only reductive reaction of the cycle:

This process is readily reversible in the light. The enzyme itself is activated by light via the so-called "light-modulation system," specifically the ferredoxin/thioredoxin system. Phosphoglyceraldehyde easily isomerizes into dihydroxyacetone phosphate (DHAP) with the participation of the enzyme Triosephosphate isomerase:

In the Calvin cycle, a significant portion of triose phosphates (GAP, DHAP) is utilized in the form of DHAP; consequently, at equilibrium in this reaction, 95% of all triose phosphates exist as DHAP. Phosphoglyceraldehyde is a phosphorylated sugar derivative containing only three carbon atoms, whereas the simplest plant CARBOHYDRATES occur as hexoses. To synthesize them, two molecules of GAP or other trioses (GAP, DHAP) must join HEAD-to-head. This reaction is catalyzed by the enzyme fructose-bisphosphate aldolase:

As a rule, during each turn of the Calvin cycle, two GAP molecules are converted into DHAP, and then, in the presence of aldolase, GAP and DHAP condense to form fructose-1,6-bisphosphate. Following dephosphorylation, fructose-6-phosphate can be exported from the cycle and used for the synthesis of sucrose or other polysugars. Furthermore, through respiratory pathways, they become integrated into the carbon skeletons of numerous other organic substances.

For photosynthetic CO₂ fixation to proceed without interruption, the primary acceptor must be regenerated at the same rate at which it is consumed. Therefore, a regeneration mechanism must exist. Indeed, in the Third Stage of the cycle, five triose phosphate molecules are converted into three molecules of the primary acceptor: 5C₃ → 3C₅. This is achieved through a series of isomerization and molecular rearrangement reactions. It should be noted that all these reactions occur at the same energy level without any additional ATP expenditure.

First, with the participation of transketolase, F-6-P and GAP are used to synthesize the 4-carbon erythrose-4-phosphate (E-4-P) and the 5-carbon xylulose-5-phosphate (Xu-5-P):

In this reaction, phosphoglyceraldehyde reacts with an equimolar amount of fructose-6-phosphate, resulting in The formation of equal quantities of 4- and 5-carbon sugars:

C₆ + C₃ → C₄ + C₅

Next, erythrose-4-phosphate reacts with an equal amount of DHAP through the action of aldolase. The product of this aldol Condensation is sedoheptulose-1,7-bisphosphate (S-1,7-BP):

Then SBP, mediated by SBP-phosphatase, is dephosphorylated into sedoheptulose-7-phosphate (Su-7-P):

This reaction is considered the rate-limiting step of this PCR cycle, and therefore it plays a key role in its regulation.

The next reaction again involves transketolase, in which sedoheptulose-7-phos- phate and phosphoglyceraldehyde yield xylulose-5-phosphate and ribose-5-phosphate:

In this reaction, transketolase transfers a two-carbon fragment to phosphoglyceraldehyde, but this time from sedoheptulose-7-phosphate rather than fructose-6-phosphate, which explains why two pentoses are formed in this case:

This indicates that a phosphoglyceraldehyde molecule can be utilized in the Calvin cycle in four different ways. This completes the conversion of 5 PGA molecules into 3 pentose molecules.

Finally, two molecules of xylulose-5-phosphate (Xlu-5-P) are converted into ribulose bisphosphate with the participation of the enzyme ribose phosphate epimerase:

This enzyme essentially catalyzes an isomerization reaction, but the substrate and product are epimers that differ from each other only in the orientation of the hydrogen atom and the hydroxyl group at the third carbon atom.

The ribose-5-phosphate synthesized during the cycle is also isomerized to ribulose-5-phosphate by the enzyme ribulose phosphate isomerase:

Thus, in The final stage, the enzyme phosphoribulokinase ensures the phosphorylation of ribulose-5-phosphate using ATP from the light-dependent stage, regenerating the primary carbon dioxide acceptor, ribulose-1,5-bisphosphate. This reaction brings the Calvin cycle to a close:

Autocatalysis. A reaction that produces more substrate than it consumes is called autocatalytic, because its rate increases with a rise in Substrate Concentration. For every three CO₂ molecules incorporated into the PCR cycle, one molecule of the product—triose phosphate—is formed. It can be converted into starch, sucrose, etc., as well as used to regenerate the CO₂ acceptor, thereby stimulating the formation of intermediate products. In other words, the cycle Functions in such a way that it produces more acceptor than it consumes. If the cycle regenerated only as much substrate as it used, the plant Organism would lack The ability to grow and adapt to more favorable conditions by increasing the intensity of photosynthesis. Today, green plants are unique in their ability to use the PCR cycle to convert photosynthetic products into additional substrate.

Thus, during the cycle, 6 CO₂ are converted into fructose-6-phosphate, consuming 12 NADPH and 18 ATP in the process. Assimilating one molecule of CO₂ requires 2 NADPH and 3 ATP. Actual energy costs are somewhat higher due to photorespiration, during which a certain portion of it is lost. The overall net equation of the dark phase is as follows:

The resulting triose phosphate (PGA) enters the next stage of forming the final products of photosynthesis. From this, the Energy balance of the Calvin cycle can be calculated. Reducing six carbon dioxide molecules to the carbohydrate level (C₆H₁₂O₆) requires 18 ATP molecules and 12 NADPH₂. Accordingly, reducing a single CO₂ molecule to the carbohydrate level requires three ATP molecules and two NADPH₂. We know that the formation of two NADPH₂ molecules and two ATP molecules requires 8 light quanta. The remaining ATP is produced via cyclic Photophosphorylation. Consequently, reducing a single carbon dioxide molecule to the carbohydrate level requires a minimum of 8–9 quanta. The energy of red light quanta is 168 kJ per mole. Thus, when using red light rays, approximately 1340–1508 kJ is expended to reduce one CO₂ molecule to the carbohydrate level. Of this energy, 478 kJ is stored in one-sixth of a mole of hexose. The photosynthetic efficiency in this case is approximately 30–35%. However, under natural conditions, the light utilization coefficient is significantly lower. The pathway of carbon in photosynthesis established by Calvin is the primary one, although deviations from this pathway do exist.