We have seen that NADPH and ATP are produced in the light phase of photosynthesis. The next phase of photosynthesis involves the fixation of CO2 into carbohydrates. Although many textbooks state that glucose (C6H12O6) is the major product of photosynthesis, the actual carbohydrate endproducts are sucrose, paramylon, starch, etc. The fixation of
CO2 takes place during the light independent phase using the assimilatory power of NADPH and ATP in the chloroplast stroma (eukaryotic algae) or in the cytoplasm (prokaryotic algae). The lightindependent reactions do not occur in the dark; rather they occur simultaneously with the light reactions. However, light is not directly involved. The light-independent reactions are commonly
referred to as the Calvin Benson Bassham cycle (CBB cycle) after the pioneering work of its discoverers.
The first metabolite was a 3-carbon organic acid known as 3-phosphoglycerate (3-PG). For this reason, the pathway of carbon fixation in algae and most plants is referred to as C3 photosynthesis. As the first product was a C3 acid, Calvin hypothesized that the CO2 acceptor would be a C2 compound. However, no such C2 substrate was found. Rather, it was realized that the CO2 acceptor was a C5 compound, ribulose 1,5-bisphosphate (RuBP), and that the product of carboxylation was two molecules of 3-PG. This crucial insight allowed the pathway of carbon flow to be determined.
While the CBB cycle involves a total of 13 individual enzymatic reactions, only two enzymes are unique to this pathway: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphorybulokinase (PRK). All other enzymes involved also perform functions in heterotrophic metabolism. PRK catalyzes the phosphorylation of ribulose-monophosphate to ribulose-1,5- bisphosphate (RUBP). RUBP in turn is the substrate for RuBisCO, which catalyzes the actual carbon fixation reaction.
As a result, the RuBisCO enzyme alone represents the most important pathway by which inorganic carbon enters the biosphere. It has also been described as the most abundant protein on Earth. It is thought that as much as 95% of all carbon fixations by C3 organisms (that includes all phytoplankton) occur through RuBisCO.
RuBisCO is known to catalyze at least two reactions: the reductive carboxylation of ribulose 1,5-bisphosphate (RuBP) to form two molecules of 3-phosphoglycerate and the oxygenation of RuBP to form one molecule of 3-PG and one molecule of 2-phosphoglycolate.
The oxygenation of RuBP is commonly referred to as photorespiration and has traditionally been seen as a wasteful process, in particular because the regeneration of RuBP in photorespiration leads to the evolution of CO2 and requires free energy in the form of ATP. In addition RuBisCO suffers from several other inefficiencies.
Both reactions (carboxylation and oxygenation) occur in the same active site and compete, making the enzyme extremely sensitive to local partial pressures of CO2 and O2. RuBisCO makes up 20–50% of the protein in chloroplasts. It acts very slowly, catalyzing three molecules per second. This is comparable to 1000 per second typical for enzymatic reactions. Large quantities are needed to compensate for its slow speed. It may be the most abundant protein on Earth. Lastly, RuBisCO rarely performs its function at a maximum rate (Kmax), since the partial pressure of CO2 in the vicinity of the enzyme is often smaller than even its Michaelis-Menten half-saturation constant (Km). RuBisCO has been shown to occur in two distinct forms in nature termed forms I and II, respectively. Form I of the enzyme is an assemblage of eight 55 kDa large subunits (rbcL) and eight 15 kDa small subunits (rbcS). These subunits assemble into a 560 kDa hexadecameric protein-complex designated as L8S8. Most photosynthetic prokaryotes that depend on the CBB-cycle for carbon assimilation and all eukaryotic algae express a form I type RuBisCO. The exception to this rule is several marine dinoflagellates, which apparently contain a nuclear encoded form II of RuBisCO. Form II of RuBisCO is a dimer of large subunits (L2) and is otherwise found in many photosynthetic and chemoautotrophic bacteria. Phylogenetic analysis of large number of form I rbcL DNA sequences revealed the division of form I into four major clades referred to as IA, IB, IC, and ID. Form IA is commonly found in nitrifying and sulfur oxidizing chemoautotrophic bacteria as well as some marine Synechococcus (marine type A) and all Prochlorococcus strains sequenced to date. All other cyanobacteria as well as all green algae
possess a form IB type enzyme. Form IC of rbcL is expressed by some photosynthetic bacteria such as hydrogen oxidizers. Form ID encompasses a diverse group of eukaryotic lineages including essentially all chromophytic, eukaryotic algae such as Phaeophyceae, Rhodophyta, Bacillariophyceae, and Raphidophyceae.
The phylogeny of RuBisCO displays several interesting incongruencies with phylogenies derived from ribosomal DNA sequences. This has lead to the speculation that over evolutionary history numerous lateral gene transfers may have occurred, transferring RuBisCO among divergent lineages. For example the dinoflagellate Gonyaulax polyhedra contains a form II RuBisCO most similar to sequences found in proteobacteria. Within the form I clade as many as six lateral transfers have been suggested to explain the unusual phylogeny observed among the cyanobacteria, proteobacteria, and plastids. Some bacteria may have acquired a green-like cyanobacterial gene, while marine Synechococcus and Prochlorococcus almost certainly obtained their RuBisCO genes from a purple bacterium.
Three-dimensional structures of the RuBisCO enzyme are now known for a number of species, including Synechococcus and most recently the green alga Chlamydomonas reinhardtii. On the basis of these data and other studies it is now believed that the primary catalytic structure of RuBisCO is a dimer of two large subunits (L2). In form I RuBisCO four L2 dimers are cemented to form L8S8 hexadecameric superstructure whereby the major contacts between the L2 dimers are mediated by the small subunits. A Mg2+ cofactor as well as the carbamylation of Lys201 is required for the activity of the enzyme. A loop in the beta barrel and two other elements of the large subunit, one in the N and one in the C terminus of the protein form the active site in Synechococcus. Small
subunits apparently do not contribute to the formation of the active site.