Biology Notes on Autotrophic Metabolism

The below mentioned article provides notes on autotrophic metabolism.

Autotrophy generally means the ability of organisms to use inorganic carbon in the form of CO₂ as the sole source of carbon for synthesizing organic compounds necessary to build cell components. This ability is also sometimes called carbon-autotrophy to distinguish the ability of some organisms to use molecular nitrogen as the sole source of nitrogen.

Such organisms are referred to sometimes as nitrogen autotrophs. However, the term autotrophy is commonly used for carbon autotrophy. Primarily, this property is present in plants, algae and phototrophic bacteria including cyanobacteria.

Besides these organisms, all of which carry out photosynthesis, there are several groups of non-photosynthetic bacteria which can grow using CO₂ as sole source of carbon by virtue of their ability to oxidize inorganic compounds. Such organisms are chemoautotrophic or chemolithotrophic.

CO₂ is the end-product of aerobic respiration, a process which releases the energy of respiratory substrates. Carbon dioxide is, therefore, poor in energy content. In autotrophic metabolism, this energy-poor compound is used to build organic molecules which are much richer in energy content.

It is easily understandable therefore, that conversion of CO₂ to organic compound requires input of energy from an external source. The ultimate source in case of photosynthesis is radiant energy and in case of chemolithotrophy is oxidation energy of inorganic chemical compounds. In either case, the immediate source of energy for driving the endergonic reaction involved in conversion of CO₂ to organic compounds is ATP.

In photosynthesis, ATP is generated with the help of photosynthetic pigments through a process known as photophosphorylation. In chemoautotrophy, the energy of oxidation of inorganic compounds is channelized into the respiratory chain for ATP synthesis by oxidative phosphorylation.

Besides ATP, reduction of CO₂ to organic molecules e.g. glucose, requires an external source of hydrogen and electrons. In green plant photosynthesis, water acts as the terminal hydrogen donor or reductant, whereby water is oxidized to molecular oxygen which is released as by-product. In case of phototrophic anoxygenic bacteria, inorganic or organic molecules, such as H₂, H₂S, S₂O₃^— Alcohols etc. act as the external H-donor. Hydrogen and electrons donated by these external donors are transferred to NAD or NADP producing NADH₂ or NADPH₂ which are the biochemically utilizable reductants used by the cells for reduction of CO₂ to organic compounds. NADH₂ and NADPH₂ constitute the reducing force of CO₂-reduction.

Thus, autotrophic metabolism can be considered to consist of two sets of reactions. In one set, ATP and reducing force are generated and, in the other set of reactions they are utilized for reduction of CO₂ to organic compounds. The first sets of reactions are different in phototrophic and non- phototrophic autotrophs.

But the second set of reactions is common between the two groups. In majority of autotrophs, the reactions involved in reduction of CO₂ proceed via a cyclic pathway, known as the reductive pentose phosphate pathway or, more commonly, as the Calvin-Benson cycle, although other pathways are also known to operate in some organisms, both in the phototrophic green plants and bacteria. Reduction of CO₂ to yield organic compounds is commonly known as CO₂-fixation.

It is evident, therefore, that there is a close parallelism between the phototrophic and non- phototrophic autotrophic metabolism. While all phototrophic organisms carry out some type of photosynthesis, the non-green autotrophs carry out an allied process which was designated by Winogradsky as chemosynthesis.

The parallelism between these two modes of metabolism is schematically represented in Fig. 8.59:

(i) Chemoautotrophy:

Chemoautotrophy or chemolithotrophy is a mode of metabolism restricted to several specialized groups of non-photosynthetic autotrophic bacteria. Some of these bacteria are capable of growing both chemoorganotrophically and chemo-auto-trophically i.e. they are facultative autotrophs. Examples of this type are Alcaligenes eutrophus and Nitrococcus oceanus.

Other chemoautotrophic bacteria are obligate in nature, because they are unable to utilize organic compounds as carbon source. The best examples of this type are the species of the genera Nitrosomonas and Thiobacillus. Some species of Thiobacillus are, however, facultative.

Depending on the oxidisable inorganic substrate, the chemoautotrophic bacteria can be distinguished into the following groups: the nitrifying bacteria, the sulfur oxidising bacteria, the hydrogen oxidizing bacteria, the iron oxidising bacteria and the carbon monoxide bacteria.

All of them are strictly aerobic, except some which are facultative anaerobes and can grow in absence of free oxygen utilizing nitrate as an alternative electron acceptor (nitrate respiration). Examples of this latter group are Paracoccus denitrificans and Thiobacillus denitrificans.

(a) Nitrifying bacteria and nitrification:

Nitrification is a natural process carried out by the nitrifying bacteria occurring in soil and aquatic bodies. It involves oxidation of ammonia liberated by decomposition of nitrogenous organic matter, like proteins, nucleic acids, urea etc. The oxidation takes place in two steps — ammonia to nitrous acid and nitrous acid to nitric acid.

The acids react with metal ions to produce the corresponding salts, viz. nitrite and nitrate. Nitrate acts as the main nitrogen source of plants, although many plants can also use ammonium ion as nitrogen source. The negatively charged nitrate ions (N0₃^–) have greater diffusibility in soil compared to that of positively charged ammonium ion (NH₄⁺), because the soil particles (clay), which are also negatively charged, tend to bind positively charged ammonium ions more tightly preventing their diffusion.

The two-step nitrification process is carried out by two different groups of bacteria. The first step involving oxidation of ammonia to nitrous acid is called nitrosification and the organisms are accordingly known as nitrosifying bacteria.

The most important representative of this group is Nitrosomonas. The members of this genus are highly aerobic and strictly autotrophic. They are incapable of utilizing any organic compound and can grow only in purely inorganic salts medium. The energy-yielding oxidation reaction of these bacteria can be represented as 2 NH4 + 3O₂ —> 2 NO^–₂ + 4H⁺ + 2H₂O. The organisms carrying out this reaction possess the enzyme ammonia dehydrogenase.

The second step of nitrification involves oxidation if nitrous acid to nitric acid and the organisms are known as nitrifying bacteria. The most well-known genus of the group is Nitrococcus. The reaction occurring in these bacteria can be represented as 2NO₂^– + O₂ —> 2NO^–₃. The enzyme catalyzing this reaction is nitrous acid dehydrogenase. In contrast to the obligately autotrophic Nitrosomonas species, Nitrobacter can grow both auto-trophically as well as using acetate as carbon source. Thus, Nitrobacter species are facultative chemoautotrophs.

The organisms of both groups are capable of generating ATP by oxidative phosphorylation in course of electron transport through the cytochrome system of the respiratory chain and the final electron acceptor is oxygen. ATP generated in this way is utilized for CO₂-fixation by the Calvin- Benson cycle.

However, the question of how NADH₂ is generated in these bacteria — particularly in the nitrite-oxidizing bacteria — is not easily explainable. This is because the oxidisable substrate, nitrite (NO^–₂) has a more positive (or less negative) redox potential than that of NAD. This does not permit spontaneous flow of electrons from NO₂ upstream to NAD, unless energy is expended.

It has been suggested, therefore, that part of the ATP generated by oxidative phosphorylation is spent for driving electrons from nitrite to NAD through a reverse electron transport i.e. from a less negative redox potential to a more negative one. Thus, generation of the reducing force (NADH₂) in these bacteria appears to be an energy-consuming process.

(b) Sulfur oxidizing bacteria:

Oxidation of elemental sulfur (S⁰) and various reduced sulfur compounds, like sulfide (S^2-), thiosulfate (S₂O²₃^–) etc. takes place in soil and aquatic bodies mediated by a great variety of bacteria including both eubacteria and archaebacteria. However, all of them are not chemoautotrophic i.e. they cannot utilize the oxidation energy for autotrophic growth. The best-known among the sulfur-oxidising eubacteria are the members of the genus Thiobacillus. Some species, like T. thiooxidans, T. thioparus and T. denitrificans are obligately chemoautotrophic, while other species, like T. novellus or T. intermedius are facultative and can utilize also organic compounds by heterotrophic pathways. Thiobacilli in general are strongly aerobic, but T. denitrificans can also grow anaerobically carrying out nitrate respiration.

Among sulfur-oxidising archaebacteria, Sulfolobus, an aerobic thermophilic as well as acidophilic organism (optimum temperature 75°C and optimum pH 1.5-3.5) is a facultative chemoautotroph. Another one, Thermoproteus is an anaerobic thermophile (70°-90°C) which can grow facultatively as an autotroph oxidizing either sulfur or hydrogen.

Some eubacteria, designated as filamentous sulfur-oxidising bacteria, belonging to the genera Beggiatoa, Thiothrix etc. are able to oxidize sulfide (H₂S) to elemental sulfur (S⁰). However, their ability to use the oxidation energy for CO₂-fixation remains controversial. The anoxygenic sulfur purple and green bacteria, like Chromatium, Chlorobium etc. are also able to oxidize sulfide to sulfur. They use sulfide as hydrogen donor in anoxygenic photosynthesis, just as green plants use water.

A remarkably large spherical bacterium (750 μm in diameter) was discovered in 1999 in the coastal sediments collected from a depth of more than 100 meter near Namibia (South West Africa). The organism has been named as Thiomargarita namibiensis. It is capable of growing autotrophically utilizing oxidation of hydrogen sulfide, and nitrate is used as terminal electron acceptor (nitrate respiration). The organism oxidizes H₂S to S° and the sulfur globules are deposited intracellularly. This happens also in case of Beggiatoa, Thiothrix, Chromatium etc.

Thiobacilli oxidize elemental sulfur or sulfur compounds to sulfuric acid.

The reactions can be represented as:

Hydrogen and electrons are transferred from the substrates in course of oxidation to specific acceptors which are then end-oxidised via electron transport system to produce ATP. The production of NADH₂ is faced with the same problem as with nitrite oxidation, because electrons liberated by oxidation of sulfur, sulfide or thiosulfate cannot be transferred directly to NAD as their redox-potentials are more positive than that of NAD. Therefore, NADH₂ is presumably produced by an ATP-consuming reverse electron flow in case of sulfur-oxidising bacteria also.

Thiobacilli fix CO₂ via Calvin-Benson cycle in which ATP and NADH₂ provide energy and reducing force, respectively. That this cycle operates in chemoautotrophic bacteria was first demonstrated in Thiobacillus denitrificans.

Thiobacillus thiooxidans is outstanding for its very high acid tolerance. It can grow even at pH 0. This property has been practically utilized for reclamation of alkaline soils. Soils having excess of Ca-carbonate or oxide are treated with elemental sulfur in powder form which stimulates growth of T. thiooxidans in soil. The bacteria produce sulfuric acid and converts calcium salts into CaSO₄ which is more soluble than the carbonate. By leaching, the sulfate can be gradually removed.

Thiobacilli producing plenty of sulfuric acid are also used for extraction of metals by the leaching process. For example, T. ferrooxidans, which is highly resistant to copper, is utilized for extraction of copper from low-grade ores. Such ores are mixed with iron-pyrites (FeS). Thiobacilli grow and produce sulfuric acid from the sulfide which leaches out copper as copper sulfate in the leachate from where copper in recovered.

(c) Hydrogen oxidizing bacteria:

A diverse group of bacteria, both eubacteria and archaebacteria possess the ability to oxidize molecular hydrogen. Many of them can use the energy of hydrogen oxidation for autotrophic growth. However, most of these bacteria are facultative i.e. they can also grow as heterotrophs using a variety of organic compounds as carbon and energy source. Some like Paracoccus denitrificans are mixotrophic a type of metabolism in which part of the carbon requirement is fulfilled by CO₂ and a part by some organic compound.

Among the well-characterized hydrogen oxidizing bacteria are Alcaligenes (formerly Hydrogenomonas) eutrophus, A. ruhlandii, Pseudomonas facilis, Xanthobacter autotrophicum, Azospirillum lipoferum, Nocardia opaca, Paracoccus denitrificans etc. All of them are facultative chemoautotrophs. They can grow in an inorganic salts medium when the atmosphere contains about 70% H₂, 20% O₂ and 10% CO₂.

Some non-pathogenic mycobacteria can also grow as facultative autotrophs. A number of extremophilic archaebacteria can grow as hydrogen-bacteria e.g. Thermoproteus (facultative), Pyrodictium (anaerobic, obligately chemoautotrophic) and Methanococcus jannaschii (obligate).

Under autotrophic conditions i.e. in absence of any organic carbon source and in presence of an atmosphere containing H₂, O₂ and CO₂, the hydrogen-oxidising enzyme, hydrogenase, is synthesized in the hydrogen bacteria. The energy-yielding hydrogen oxidation catalysed by this enzyme can be represented as 2H₂ + O₂ —> 2H₂O (-∆G =112 Kcal).

The enzyme hydrogenase performs two important functions. Firstly, it can transfer hydrogen directly to NAD producing NADH₂ which is the reducing force required for CO₂ reduction via Calvin-Benson cycle. Secondly, the enzyme can transfer electrons to the electron transport chain to generate ATP by oxidative phosphorylation. Hydrogenase is a nickel containing enzyme and the bacteria require Ni²⁺ for autotrophic growth, but not when they grow in organic media carrying out heterotrophic metabolism. Obviously, hydrogenase is synthesized only under autotrophic conditions.

In some hydrogen-bacteria the hydrogenase is of two types — one is in soluble state (cytoplasmic) and the other is particle-bound (membrane associated). In such organisms the two types perform separate functions. While the particulate type is involved in supplying electrons donated by hydrogen to ubiquinone of the respiratory chain, the soluble type transfers hydrogen and electrons for reduction of NAD to NADH₂.

The functions of these two types of hydrogenase are diagrammatically shown in Fig. 8.60:

Some hydrogen bacteria have both membrane-bound as well as soluble hydrogenases, e.g. Alcaligenes eutrophus, A. ruhlandii, Pseudomonas saccharophila etc. Some others have only one type e.g. Nocardia opaca and N. autotrophica. They contain only the soluble NAD-reducing hydrogenase. On the other hand Paracoccus denitrificans, Aquaspirillum autotrophicum, Xanthobacter autotrophicus, as well as several pseudomonads have only the membrane-bound type.

(d) Carboxido bacteria:

A group of aerobic bacteria are capable of chemoautotrophic growth by virtue of their ability to oxidize carbon monoxide CO to CO₂. These organisms can also grow as hydrogen bacteria. Carboxidobacteria can grow in a mineral-salt medium with an atmosphere containing CO and O₂. The oxidation reaction is catalysed by the enzyme carbon monoxide oxidoreductase. It can be represented as CO + H₂O CO₂ + 2H⁺ + 2e^–.

Carboxidobacteria belong to the genera Alcaligenes, Pseudomonas etc. All of them have a membrane-bound hydrogenase, while the CO-oxidising enzyme is a soluble cytoplasmic protein.

(e) Iron-oxidising bacteria:

The only iron (Fe²⁺) oxidizing bacterium that is definitely known to be capable of chemoautotrophic growth is Ferrobacillus ferro-oxidans (= Thiobacillus ferrooxidans). It is closely related to Thiobacillus thiooxidans. The organism is able to oxidize Fe²⁺ to Fe³⁺ vigorously in an acidic environment (pH 3.0) according to the following reaction. 4Fe²⁺ + 4H⁺ + O₂ —> 4 Fe³⁺ + 2H₂O (-∆G = 40 Kcal). The oxidation energy is utilized for fixation of CO₂. For assimilation of one μM of CO₂, 50 μM of Fe²⁺ is oxidized to Fe³⁺.

Ferro-bacilli can also oxidize elemental sulfur (S⁰) to sulphate like the sulfur-oxidising bacteria. This property has been commercially exploited for removal of sulfur from iron ore which improves the quality of the ore and makes it more suitable for extraction of metallic iron.

Several other bacteria, like Gallionella ferruginea, a stalked bacterium and Leptothrix ochracea can oxidize ferrous salts present in the water in which they grow to ferric oxide. The insoluble ferric oxide is deposited on their cell wall as incrustation. However, their ability of chemoautotrophic growth at the expense of the oxidation of ferrous iron to ferric iron has not been decisively proved.

(ii) Photo trophy:

Photo trophy refers to an autotrophic mode of metabolism in which organisms are able to harness light energy with the help of photosynthetic pigments and convert it to chemical bond energy in the form of ATP (photophosphorylation). Phototrophic organisms, like the chemoautotrophs, are able to form organic compounds from carbon dioxide, generally through the Calvin-Benson cycle. The two processes i.e. generation of ATP by photophosphorylation and carbon dioxide fixation, together constitute photosynthesis.

Primarily, photosynthesis is a property of plants and different groups of algae and some chlorophyllous protozoans. All of them, including the cyanobacteria (which are also known as blue- green algae), carry out photosynthesis with oxygen as by-product. Therefore, the type of photosynthesis is known as oxygenic. There are also some bacteria which contain bacteriochlorophylls and can carry out photosynthesis, but without oxygen evolution (anoxygenie).

In a broad sense, photosynthesis can be defined as a process in which light energy is used to promote cellular energy-requiring biochemical events. The capacity of photosynthesis is attributable to the presence of special pigments, called chlorophylls and carotenoids which are associated with lipoproteins to form complexes.

Photosynthesis includes two closely linked, but distinct processes. The first of these comprises the reactions by which light energy is absorbed by the photosynthetic pigments and transformed into chemical bond energy. These reactions are photochemical in nature and are known as light reactions. The second process includes enzyme-catalysed biochemical reactions involving CO₂-fixation in which light has no direct role.

These reactions are called dark-reactions. A notable feature of photochemical reactions which distinguishes them from chemical or biochemical reactions is that the reaction velocity is independent of temperature i.e. the reaction proceeds with the same velocity at, say, zero degree and 30°C. In case of chemical and biochemical reactions, the reaction velocity typically doubles with every 10°C rise of temperature within limits. In enzyme-catalysed reactions, the enzyme protein generally loses catalytic activity above 35° to 40°C.

The usual products of light reactions are ATP and NADH₂ or NADPH₂. These products are used in the dark reactions for synthesis of sugars or other organic compounds from CO₂. The dark reactions are common to both photoautotrophs and chemoautotrophs. The two groups differ mainly on the mode of generation of ATP and reducing force (NADH₂/ NADPH₂).

(a) Photosynthetic light reactions:

The first step in photosynthesis is the absorption of photons by a series of light harvesting pigments in pigment-protein complexes. These are called antenna-complexes, because they collect light of different wave-lengths. For example, the different chlorophylls generally absorb light of longer wavelengths extending up to infra-red regions by some bacteriochlorophylls.

On the other hand, carotenoids absorb light of shorter wavelengths, generally in the yellow region of the spectrum. The light energy absorbed by the antenna system is transmitted to a special photo-reactive centre, known as the reaction centre which is also a pigment-protein complex located in the photosynthetic lamellae.

The lamellae are part of chloroplasts in case of eukaryotic organisms, and in prokaryotic organisms they are the intracellular membrane system produced by invagination of the cytoplasmic membrane (chromatophores).

The reaction centre pigment complex — on being excited by energy transferred from the antenna pigments — ejects energy-rich electrons which are accepted by the primary electron acceptor, ferredoxin. Electrons from ferredoxin are then transferred to the secondary electron acceptors at a velocity which is faster than that of electron transfer from the reaction centre pigment to ferredoxin.

As a result, the reaction centre remains positively charged (due to loss of electrons). Appropriate positioning of secondary electron acceptors leads to an electron transport in one direction across the membrane and proton transport in an opposite direction with the consequent generation of an electric field (due to charge separation). The charge separation is utilized for ATP generation by the chemiosmotic mechanism.

The overall electron flow resulting in ATP generation in photosynthesis can be of two main types, — cyclic and non-cyclic. In the cyclic type, the high-energy electrons ejected by the reaction centre pigment flow through a series of electron acceptors from a higher energy level to a gradually lower energy level and return to the reaction centre, forming thereby a close circuit. The loss of energy of electrons in this cyclic path is utilized for phosphorylation of ADP to ATP. The only product of cyclic path is ATP. No NADH₂ is produced.

In the non-cyclic pathway of photosynthetic electron-transport, electrons ejected by the reaction centre pigment complex and accepted by ferredoxin are used for reduction of NAD/NADP. It becomes necessary, therefore, to draw electrons from an exogenous source, so that the reaction centre can be re-oxidised to its ground state. In case of green plants and cyanobacteria, water acts as the electron donor. Water is photolysed by chlorophyll to yield H⁺ and (OH)^–.

Protons are used for reduction of NADP, while electrons of (OH)^– are passed on to the positively charged reaction centre through cytochromes, and molecular oxygen (O₂) is evolved. In green plant photosynthesis two light reactions occur, Photosystem I and Photosystem II. The pigment complexes of these two systems are called P700 and P680, respectively.

In anoxygenic bacterial photosynthesis the situation is somewhat different. Firstly, in bacteria there is only one photosystem which is Photosystem I. Photosystem II, which is involved in oxygen evolution through photolysis of water, is absent. In bacterial non-cyclic electron transport, the exogenous electron donor may be H₂, H₂S, S^O, S₂O₃^2- or even organic compounds.

The cyclic pathway of electron transport is diagrammatically shown in Fig. 8.61:

The non-cyclic pathway of green plant photosynthesis is shown in Fig. 8.62 and a tentative non- cyclic pathway of anoxygenic photosynthesis is represented in Fig. 8.63 A and 63 B:

In the anoxygenic photosynthetic bacteria, ATP is generated by cyclic photo-phorylation. The mode of production of the reducing force, which is NADH₂ (not NADPH₂ as in green plants and cyanobacteria), is apparently variable in different groups and depends on the exogenous reductant used by the organism.

However, the non-cyclic pathways operating in bacteria are not so well understood. It has been suggested that NADH₂ production in photosynthetic bacteria may take place at least in three different ways. Firstly, when hydrogen acts as an exogenous electron donor, NAD can be directly reduced to NADH₂:

Another way may by reverse electron flow as it occurs in the nitrifying bacteria. The reverse electron flow requires input of ATP. It may occur when H2S, SO or organic compounds are used as exogenous reductants by purple sulfur and purple non-sulfur bacteria.

A third way of NADH₂ production appears to occur in green sulfur bacteria. These bacteria are able to utilize the light energy to transfer electrons from donors like H₂S, S₂0₃^2- etc. to NAD via suitable electron transport carriers as shown in Fig. 8.63 (B).

In summarizing the light reactions of oxygenic and anoxygenic photosynthesis, it may be observed that in oxygenic photosynthesis, both ATP and NADPH₂ are produced through two light reactions using water as exogenous reductant. In contrast, in the anoxygenic type only one light reaction produces ATP by a cyclic electron transport chain. The mode of reduction of NAD to NADH₂ may be different in different major groups of bacteria. As exogenous hydrogen donor, H₂, H₂S, elemental sulfur, thiosulfate or even organic compounds may be used for reduction of NAD.

(b) Photo-synthetic dark reactions:

The products of light reactions, ATP and NADPH₂ or NADH₂ are utilized in the dark reactions for reduction of CO₂ to produce sugars or other organic compounds. Most of the autotrophic organisms, including the chemoautotrophs carry out these reactions via the Calvin-Benson cycle. The green sulfur bacteria are exceptions in this regard.

The dark reactions are purely biochemical in nature catalysed by different enzymes without any direct involvement of light. The overall stoichiometry of CO₂-fixation by the Calvin-Benson cycle is given by the equation

6CO₂ + 18ATP + 12NAD (P)H₂ + 12H₂O C₆H₁₂O₆ + 18ADP + 18Pi + 12NAD(P).

In the Calvin-Benson cycle, the acceptor of CO₂ is ribulose 1,5-bis phosphate (RuBP) and the primary product of photosynthesis is 3-phosphoglyceric acid (3-PGA). This product is reduced to triose phosphates — di-hyceraldehyde phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Part of these triose phosphates produce hexose phosphate which is the final product of photosynthesis and part is used to regenerate the acceptor of CO₂ i.e. RuBP.

The cycle has been represented in a very simplified manner showing the stoichiometry of the above equation in Fig. 8.64:

Among the enzymes of the calvinbenson cycle, only two are unique to this pathway, while the rest are present also in other pathways like EMP and PPC. These two enzymes are ribulose bisphosphate carboxylase oxygenase (Rubisco) and ribulose 5-phosphate kinase or phosphoribulokinase.

The reactions catalyzed by these enzymes are:

As each complete turn of the cycle incorporates one CO₂ molecule into RuBP, six CO₂ molecules are needed to synthesize one hexose molecules. This means the cycle must operate six times to produce a single hexose molecule.

The primary product 3-PGA is phosphorylated with ATP and 3-PGA kinase to DPGA (1,3-di-phosphoglyceric acid) which is then reduced to GAP by an NAD (P) H₂ linked GAP- dehydrogenase. A portion of GAP is utilized for formation of hexose via condensation of GAP and DHAP by aldolase to produce FBP, fructose 6-phosphate and finally glucose.

All these reactions are common with those of EMP, but the reactions of Calvin-Benson cycle run in reverse direction. The rest of GAP is utilized for regeneration of RuBP for starting another turn of the cycle. Formation of RuBP from Ru5-(P) takes place by the phosphoribulokinase reaction.

Regeneration of Ru5-(P) from GAP takes place mainly by several transfer reactions, catalysed by the transaldolases and transketolases, as well as by some epimerases and isomerases. These enzymes are common between the PPC and the Calvin-Benson cycle.

The reactions of the cycle are shown in Fig. 8.65:

Formation of ribulose 5-phosphate in the cycle from glyceraldehyde 3-phosphate takes place through several transfer reactions:

1. Fructose 6-phosphate (F6P) + glyceraldehyde 3-phosphate —> xylulose 5-phosphate (Xu5P) + Erythrose 4-phosphate (E4P).

The reaction is catalysed by the TPP-linked transketolase which transfers a two-carbon group from fructose 6-phosphate to glyceraldehyde 3-phosphate producing a four-carbon sugar (erythrose 4-phosphate) and a five-carbon sugar (xylulose 5-phosphate).

2. Dihydroxyacetone phosphate (DHAP) + Erythrose 4-phosphate (E4P) —> Sedoheptulose 1,7-diphosphate (SDP)

The reaction is catalysed by transaldelose.

3. Sedoheptulose 1,7-diphosphate (SDP) —> Sedoheptulose 7-phosphate (S7P). A phosphate group is removed through the action of a phosphatase.

4. Sedoheptulose 7-phosphate (S7P) + Glyceraldehyde 3-phosphate (GAP) —> Ribose-5 phosphate (R5P) + Xylulose 5-phosphate (Xu5P)

This reaction is also catalysed by transketolose.

5. Ribose 5-phosphate (R5P) —> Ribulose 5-phosphate (Ru5P)

The aldosugar R5P is changed into a ketosugar Ru5P through the action of an isomerase.

6. Xylulose 5-phosphate (Xu5P) —> Ribulose 5-phosphate (Ru5P)

Intra-molecular rearrangements of H and OH groups of the two keto-sugar is catalysed by an epimerase.

Finally, ribulose 5-phosphate produced in reactions (5) and (6) are phosphorylated by ATP and phosphoribulokinase to ribulose bisphosphate.

The obligately anaerobic green sulfur bacteria do not use the Calvin-Benson cycle for photosynthetic CO₂-fixation. So far as it is known, these bacteria employ some reactions which are normally involved in oxidative decarboxylation of substrates.

These reactions are normally irreversible, but the green sulfur bacteria can generate a strongly electronegative reductant by its Photosystem I which is able to force the reversal of the decarboxylation reactions leading to CO₂-fixation. This reductant may be ferredoxine.

So, in these photosynthetic bacteria, CO₂ probably enters through reversal of reactions like oxidative decarboxylation of pyruvic acid and of α-ketoglutaric acid, as shown:

Normally, in aerobic and also in many fermentative organisms, pyruvic acid produced by glucose dissimilation is decarboxylated to acetyl-CoA.

The green photosynthetic bacteria possess special capacity to run the reaction in opposite direction leading to CO₂ fixation:

This reaction occurs in the TCA cycle in aerobic organisms, where it runs in opposite direction for decarboxylation of α-ketoglutaric acid to succinyl CoA. But in green sulfur bacteria, it is employed for CO₂ incorporation and not elimination. Thus, these bacteria probably use a reductive TCA cycle for CO₂-fixation.