Photorespiration - Biochemistry

As described in my previous post (Photorespiration - Prelude), photorespiration begins when oxygen (O₂) is used as a substrate by RuBisCO instead of carbon dioxide (CO₂):

Simplified Photorespiration and Calvin Cycle. Image Source: Photorespiration - Wikipedia

Instead of generating 2 molecules of 3-phosphoglycerate when CO₂ is used as substrate, RuBisCO produces only 1 molecule of 3-phosphoglycerate plus a molecule of 2-phosphoglycolate when O₂ is employed as substrate. Because 2-phosphoglycolate is inhibitory to the activity of Calvin cycle enzymes it has to be removed and recycled back to 3-phosphoglycerate in order to avoid depleting Calvin cycle intermediates. This is accomplished by metabolizing 2 molecules of 2-phosphoglycolate (2 x 2-C) to 1 molecule of 3-phosphoglycerate (1 x 3-C) + CO₂ (1 x 1-C). 2 x 2 = 3 + 1.

This recycling of 2-phosphoglycolate is very complex, involves 3 different organelles in the plant cell (chloroplast, peroxisome and mitochondrion), involves nitrogenous metabolites (including the amino acids glycine and serine), and releases ammonia (NH₃) stoichiometrically with CO₂. This ammonia has to be in turn recycled with an energy cost of ATP and reduced ferredoxin. ATP is also required to regenerate 3-phosphoglycerate from glycerate.

Loss of CO₂, and use of energy (ATP and reduced ferredoxin) greatly reduces the efficiency of photosynthesis of C3 plants in normal air. This efficiency decreases further when plants are exposed to high temperatures and/or water deficits (drought stress). At high leaf temperatures the solubility of CO₂ in water decreases more substantially than that of O₂. Therefore photorespiration is accelerated at high leaf temperatures. During drought stress, plant leaves close their stomates preventing water loss, but also preventing CO₂ uptake. This tends to result in CO₂ depletion in the sub-stomatal intercellular spaces and leads to accelerated photorespiration.

The full cycle of photorespiration is shown below:

Photorespiration in C3-plants. How plants cope with the oxygenase reaction of RuBisCO. Image Source: Photorespiration - Wikipedia

2-Phosphoglycolate generated in the chloroplast by the oxygenase activity of RuBisCO is first dephosphorylated to glycolate. Glycolate is exported from the chloroplast to the peroxisome where it is metabolized to glyxoylate by an enzyme called glycolate oxidase. This reaction consumes further O₂ and generates a toxic molecule hydrogen peroxide (H₂O₂). The latter is detoxified by the enzyme catalase, regenerating O₂. Two molecules of glyoxylate are then converted to glycine in transamination reactions, with glutamate serving as amino (NH₂) donor for one glycine, and serine serving as amino donor for the second glycine. The resulting glycine is transported to the mitochondrion where one molecule of glycine is catabolized to ammonium (NH₄⁺) and CO₂. In this reaction, catalyzed by the glycine decarboxylase complex, the remaining portion of glycine (=CH₂) is added (together with a hydroxyl group) to a second molecule of glycine to form serine. Serine formed in the mitochondrion is then transported to the peroxisome where it is transaminated, with the amino portion serving as amino donor for the second glycine (as mentioned above), and the remaining carbon skeleton forming hydroxypyruvate. Hydroxypyruvate is then reduced to glycerate, exported from the peroxisome to the chloroplast and there phosphorylated to form 3-phosphoglycerate which can re-enter the Calvin cycle.

The CO₂ released in the mitochondrion does not have ready access to RuBisCO for re-assimilation, although it is likely that some can be recaptured in this manner (see dashed line in the image above). The ammonia (or ammonium (NH₄⁺)) concomitantly released with CO₂ in the glycine decarboxylase reaction has to be re-assimilated. This is accomplished by transport of ammonium from the mitochondrion to the chloroplast and ammonium assimilation using the glutamine synthetase : glutamate synthase cycle localized in the chloroplast (see my introductory post in this series entitled From a Bicycle Paper Round to Metabolic Cycles). While this glutamine synthetase : glutamate synthase cycle is not fully depicted in the image above, the net result of this assimilation is synthesis of glutamate from 2-oxoglutarate and NH₄⁺. This re-assimilation of ammonium consumes both ATP and reduced ferredoxin. Glutamine synthetase consumes ATP, while ferredoxin-dependent glutamate synthase consumes reduced ferredoxin.

As mentioned above, glutamate becomes the amino donor for the first glycine of the photorespiratory cycle. When glutamate serves as an amino donor in glycine synthesis, 2-oxoglutarate is regenerated. Glutamate and 2-oxoglutarate are rapidly exchanged between peroxisome and chloroplast to sustain this pathway.

This is clearly a very elaborate cycle. Some call it the photorespiratory carbon oxidation cycle, while others refer to it as the photorespiratory nitrogen cycle because of the involvement of nitrogenous solutes (glycine, serine, glutamate, glutamine and NH₄⁺). Numerous membrane-bound transport proteins (translocators) are required to facilitate metabolic shuttling between compartments.

This pathway has been verified genetically by identifying mutants of C3 angiosperms which show a phenotype of normal growth when grown in an atmosphere of elevated CO₂, but which become very sick and ultimately die when exposed to normal air. Mutants of both barley (Hordeum vulgare) and Arabidopsis thaliana which show this phenotype have been extensively characterized and have deficiencies of specific enzymes of this cycle, including:

• chloroplastic glutamine synthetase, ferredoxin-dependent glutamate synthase, 2-phosphoglycerate phosphatase and glycerate kinase

• peroxisomal serine:glyoxyate aminotransferase, glutamate:glyoxylate aminotransferase, catalase, glycolate oxidase and hydroxypyruvate reductase

• mitochondrial glycine decarboxylase/serine hydroxymethyltransferase, and

• the various transport proteins (translocators)

Many of these mutants that led to confirmation of this pathway are described in the literature cited below (see References).

The chloroplastic ferredoxin-dependent glutamate synthase and glutamine synthetase mutants appear to die from over-production of NH₄⁺ when exposed to normal air and forced to carry out photorespiration. They can survive in elevated CO₂ (when photorespiration is suppressed) because they have "back-up" glutamate synthase and glutamine synthetase genes whose gene products are adequate to cope with primary nitrogen assimilation in the absence of photorespiration. However, the relatively low levels of these enzymes are inadequate to cope with the massive amounts of NH₄⁺ released during photorespiration.

This apparently wasteful cycle, which significantly lowers the productivity of C3 plants, has become the target for genetic/metabolic engineering with the goal of improving agriculturally important C3 plant performance/yield. These metabolic engineering efforts will be described in a future post: Photorespiration - Metabolic Engineering.

Please feel free to comment or ask questions below.

References

Abadie, C., Boex-Fontvieille, E.R., Carroll, A.J., Tcherkez, G. In vivo stoichiometry of photorespiratory metabolism. Nat. Plants 2: 15220 (2016)

Armstrong, A. Photorespiration: in the balance. Nat. Plants 2: 15217 (2016)

Bauwe, H., Hagemann, M., Fernie, A.R. Photorespiration: players, partners and origin. Trends Plant Sci. 15: 330-336 (2010)

Bellasio, C. A generalized stoichiometric model of C3, C2, C2+C4, and C4 photosynthetic metabolism. J. Exp. Bot. 68: 269-282 (2017)

Hagemann, M., Bauwe, H. Photorespiration and the potential to improve photosynthesis. Curr. Opin. Chem. Biol. 35: 109-116 (2016)

Kerchev, P., Waszczak, C., Lewandowska, A., Willems, P., Shapiguzov, A., Li, Z., Alseekh, S., Mühlenbock, P., Hoeberichts, F.A., Huang, J., Van Der Kelen, K., Kangasjärvi, J., Fernie, A.R., De Smet, R., Van de Peer, Y., Messens, J., Van Breusegem, F. Lack of GLYCOLATE OXIDASE1, but not GLYCOLATE OXIDASE2, attenuates the photorespiratory phenotype of CATALASE2-deficient Arabidopsis. Plant Physiol. 171: 1704-1719 (2016)

Reumann, S., Weber, A.P. Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled--others remain. Biochim. Biophys. Acta. 1763: 1496-1510 (2006)

Walker, B.J., VanLoocke, A., Bernacchi, C.J., Ort, D.R. The costs of photorespiration to food production now and in the future. Annu. Rev. Plant Biol. 67: 107-129 (2016)