Carbon fixation

Somewhere between 3.8 and 2.3 billion years ago, the ancestors of cyanobacteria evolved oxygenic photosynthesis,[3][4] enabling the use of the abundant yet relatively oxidized molecule H₂O as an electron donor to the electron transport chain of light-catalyzed proton-pumping responsible for efficient ATP synthesis.[5][6] When this evolutionary breakthrough occurred, autotrophy (growth using inorganic carbon as the sole carbon source) is believed to have already been developed. However, the proliferation of cyanobacteria, due to their novel ability to exploit water as a source of electrons, radically altered the global environment by oxygenating the atmosphere and by achieving large fluxes of CO₂ consumption.[7]

Many photosynthetic organisms have not acquired CO₂ concentrating mechanisms (CCMs), which increase the concentration of CO₂ available to the initial carboxylase of the Calvin cycle, the enzyme RuBisCO. The benefits of a CCM include increased tolerance to low external concentrations of inorganic carbon, and reduced losses to photorespiration. CCMs can make plants more tolerant of heat and water stress.

CO₂ concentrating mechanisms use the enzyme carbonic anhydrase (CA), which catalyze both the dehydration of bicarbonate to CO₂ and the hydration of CO₂ to bicarbonate

HCO₃⁻ + H⁺ ⇌ CO₂ + H₂O

Lipid membranes are much less permeable to bicarbonate than to CO₂. To capture inorganic carbon more effectively, some plants have adapted the anaplerotic reactions

HCO₃⁻ + H⁺ + PEP → OAA + P_i

catalyzed by PEP carboxylase (PEPC), to carboxylate phosphoenolpyruvate (PEP) to oxaloacetate (OAA) which is a C₄ dicarboxylic acid.

CAM plants that use Crassulacean acid metabolism as an adaptation for arid conditions. CO₂ enters through the stomata during the night and is converted into the 4-carbon compound, malic acid, which releases CO₂ for use in the Calvin cycle during the day, when the stomata are closed. The dung jade plant (Crassula ovata) and cacti are typical of CAM plants. Sixteen thousand species of plants use CAM.[8] These plants have a carbon isotope signature of −20 to −10 ‰.[9]

C₄ plants preface the Calvin cycle with reactions that incorporate CO₂ into one of the 4-carbon compounds, malic acid or aspartic acid. C₄ plants have a distinctive internal leaf anatomy. Tropical grasses, such as sugar cane and maize are C₄ plants, but there are many broadleaf plants that are C₄. Overall, 7600 species of terrestrial plants use C₄ carbon fixation, representing around 3% of all species.[10] These plants have a carbon isotope signature of −16 to −10 ‰.[9]

The large majority of plants are C₃ plants. They are so-called to distinguish them from the CAM and C₄ plants, and because the carboxylation products of the Calvin cycle are 3-carbon compounds. They lack C₄ dicarboxylic acid cycles, and therefore have higher CO₂ compensation points than CAM or C₄ plants. C₃ plants have a carbon isotope signature of −24 to −33‰.[9]

Almost all cyanobacteria and some bacteria utilize carboxysomes to concentrate carbon dioxide. Carboxysomes are protein shells filled with the enzyme RuBisCO and a carbonic anhydrase. The carbonic anhydrase produces CO₂ from the bicarbonate that diffuses into the carboxysome. The surrounding shell provides a barrier to carbon dioxide loss, helping to increase its concentration around RuBisCO.

The reverse Krebs cycle, also known as reverse TCA cycle (rTCA) or reductive citric acid cycle, is an alternative to the standard Calvin-Benson cycle for carbon fixation. It has been found in strict anaerobic or microaerobic bacteria (as Aquificales)[11] and anaerobic archea. It was discovered by Evans, Buchanan and Arnon in 1966 working with the photosynthetic green sulfur bacterium Chlorobium limicola.[12] The cycle involves the biosynthesis of acetyl-CoA from two molecules of CO₂.[13] The key steps of the reverse Krebs cycle are:

• Oxaloacetate to malate, using NADH + H⁺

${\displaystyle {\ce {Oxaloacetate + NADH/H+ -> Malate + NAD+}}}$

• Fumarate to succinate, catalyzed by an oxidoreductase, Fumarate reductase

${\displaystyle {\ce {Fumarate + FADH2 <=> Succinate + FAD}}}$

• Succinate to succinyl-CoA, an ATP dependent step

${\displaystyle {\ce {Succinate + ATP + CoA -> Succinyl-CoA + ADP + Pi}}}$

• Succinyl-CoA to alpha-ketoglutarate, using one molecule of CO₂

${\displaystyle {\ce {Succinyl-CoA + CO2 + Fd{(red)}-> alpha-ketoglutarate + Fd{(ox)}}}}$

• Alpha-ketoglutarate to isocitrate, using NADPH + H⁺ and another molecule of CO₂

${\displaystyle {\ce {Alpha-ketoglutarate + CO2 + NAD(P)H/H+ -> Isocitrate + NAD(P)+}}}$

• Citrate converted into oxaloacetate and acetyl-CoA, this is an ATP dependent step and the key enzyme is the ATP citrate lyase

${\displaystyle {\ce {Citrate + ATP + CoA -> Oxaloacetate + Acetyl-CoA + ADP + Pi}}}$

This pathway is cyclic due to the regeneration of the oxaloacetate.[14]

The reverse Krebs cycle is used by microorganisms in anaerobic environments. In particular, it is one of the most used pathways in hydrothermal vents by the Epsilonproteobacteria.[15] This feature is very important in oceans. Without it, there would be no primary production in aphotic environments, which would lead to habitats without life. So this kind of primary production is called "dark primary production".[16]

One other important aspect is the symbiosis between Gammaproteobacteria and Riftia pachyptila. These bacteria can switch from the Calvin-Benson cycle to the rTCA cycle and vice versa in response to different concentrations of H₂S in the environment.[17]

The reductive acetyl CoA pathway (CoA) pathway, also known as the Wood-Ljungdahl pathway, was discovered by Harland G. Wood and Lars G. Ljungdahl in 1965, thanks to their studies on Clostridium thermoaceticum, a Gram positive bacterium now named Moorella thermoacetica.[18] It is an acetogen, an anaerobic bacteria that uses CO₂ as electron acceptor and carbon source, and H₂ as an electron donor to form acetic acid.[19][20][21][22] This metabolism is wide spread within the phylum Firmicutes, especially in the Clostridia.[19]

The pathway is also used by methanogens, which are mainly Euryarchaeota, and several anaerobic chemolithoautotrophs, such as sulfate-reducing bacteria and archaea. It is probably performed also by the Brocadiales, an order of Planctomycetes that oxidize ammonia in anaerobic condition.[13][23][24][25][26][27][28] Hydrogenotrophic methanogenesis, which is only found in certain archaea and accounts for 80% of global methanogenesis, is also based on the reductive acetyl CoA pathway.

The Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase is the oxygen-sensitive enzyme that permits the reduction of CO₂ to CO and the synthesis of acetyl-CoA in several reactions.[29]

One branch of this pathway, the methyl branch, is similar but non-homologous between bacteria and archaea. In this branch happens the reduction of CO₂ to a methyl residue bound to a cofactor. The intermediates are formate for bacteria and formyl-methanofuran for archaea, and also the carriers, tetrahydrofolate and tetrahydropterins respectively in bacteria and archaea, are different, such as the enzymes forming the cofactor-bound methyl group.[13]

Otherwise, the carbonyl branch is homologous between the two domains and consists of the reduction of another molecule of CO₂ to a carbonyl residue bound to an enzyme, catalyzed by the CO dehydrogenase/acetyl-CoA synthase. This key enzyme is also the catalyst for the formation of acetyl-CoA starting from the products of the previous reactions, the methyl and the carbonyl residues.[29][30]

This carbon fixation pathway requires only one molecule of ATP for the production of one molecule of pyruvate, which makes this process one of the main choice for chemolithoautotrophs limited in energy and living in anaerobic conditions.[13]

The 3-Hydroxypropionate bicycle, also known as 3-HP/malyl-CoA cycle, was discovered by Helge Holo in 1989. It's a pathway of carbon fixation and is utilized by green non-sulfur phototrophs of Chloroflexaceae family, including the maximum exponent of this family Chloroflexus auranticus by which this way was discovered and demonstrated.[31]

The 3-Hydroxipropionate bicycle is composed of two cycles and the name of this way comes from the 3-Hydroxyporopionate which corresponds to an intermediate characteristic of it.

The first cycle is a way of synthesis of glycoxilate. During this cycle two bicarbonate molecules are fixed thanks to the action of two enzymes: the Acetyl-CoA carboxylase catalyzes the carboxylation of the Acetyl-CoA to Malonyl-CoA and Propionyl-CoA carboxylase catalyses the carboxylation of Propionyl-CoA to Methylamalonyl-CoA. From this point a series of reactions lead to the formation of glycoxylate which will thus become part of the second cycle.[32][33]

In the second cycle, glycoxilate is approximately one molecule of Propionyl-CoA forming Methylamalonyl-CoA. This, in turn, is then converted through a series of reactions into Citramalyl-CoA. The Citramalyl-CoA is split into pyruvate and Acetyl-CoA thanks to the enzyme MMC lyase. At this point the pyruvate is released, while the Acetyl-CoA is reused and carboxylated again at Malonyl-coa thus reconstituting the cycle.[34]

19 are the total reactions involved in 3-Hydroxypropionate bicycle and 13 are the multifunctional enzymes used. The multifunctionality of these enzymes is an important feature of this pathway which thus allows the fixation of 3 bicarbonate molecules.[34]

It is a very expensive way: 7 ATP molecules are used for the synthesis of the new pyruvate and 3 ATP for the phosphate triose.[33]

An important characteristic of this cycle is that it allows the co-assimilation of numerous compounds making it suitable for the mixotrophic organisms.[33]

A variant of the 3-hydroxypropionate cycle was found to operate in the aerobic extreme thermoacidophile archaeon Metallosphaera sedula. This pathway is called the 3-hydroxypropionate/4-hydroxybutyrate cycle.[35]

Yet another variant of the 3-hydroxypropionate cycle is the dicarboxylate/4-hydroxybutyrate cycle. It was discovered in anaerobic archaea. It was proposed in 2008 for the hyperthermophile archeon Ignicoccus hospitalis.[36]