Ribulose-1,5-bisphosphate carboxylase-oxygenase, commonly known by the abbreviations RuBisCo, rubisco,
RuBPCase, or RuBPco, is an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
() involved in the first major step of
carbon fixation
Biological carbon fixation or сarbon assimilation is the process by which inorganic carbon (particularly in the form of carbon dioxide) is converted to organic compounds by living organisms. The compounds are then used to store energy and as ...
, a process by which atmospheric
carbon dioxide
Carbon dioxide (chemical formula ) is a chemical compound made up of molecules that each have one carbon atom covalently double bonded to two oxygen atoms. It is found in the gas state at room temperature. In the air, carbon dioxide is transpar ...
is converted by plants and other
photosynthetic
Photosynthesis is a process used by plants and other organisms to convert light energy into chemical energy that, through cellular respiration, can later be released to fuel the organism's activities. Some of this chemical energy is stored in c ...
organisms to
energy-rich molecule
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
s such as
glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
. In chemical terms, it
catalyzes the
carboxylation
Carboxylation is a chemical reaction in which a carboxylic acid is produced by treating a substrate with carbon dioxide. The opposite reaction is decarboxylation. In chemistry, the term carbonation is sometimes used synonymously with carboxylatio ...
of
ribulose-1,5-bisphosphate
Ribulose 1,5-bisphosphate (RuBP) is an organic substance that is involved in photosynthesis, notably as the principal acceptor in plants. It is a colourless anion, a double phosphate ester of the ketopentose (ketone-containing sugar with five car ...
(also known as RuBP). It is probably the most abundant enzyme on Earth.
Alternative carbon fixation pathways
RuBisCO is important
biologically
Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary i ...
because it catalyzes the primary
chemical reaction
A chemical reaction is a process that leads to the IUPAC nomenclature for organic transformations, chemical transformation of one set of chemical substances to another. Classically, chemical reactions encompass changes that only involve the pos ...
by which
inorganic carbon
Total inorganic carbon (''C''T or TIC) is the sum of the inorganic carbon species.
Carbon Chemical compound, compounds can be distinguished as either Organic compound, organic or inorganic, and Dissolved organic carbon, dissolved or Particulate ...
enters the
biosphere
The biosphere (from Greek βίος ''bíos'' "life" and σφαῖρα ''sphaira'' "sphere"), also known as the ecosphere (from Greek οἶκος ''oîkos'' "environment" and σφαῖρα), is the worldwide sum of all ecosystems. It can also be ...
. While many
autotroph
An autotroph or primary producer is an organism that produces complex organic compounds (such as carbohydrates, fats, and proteins) using carbon from simple substances such as carbon dioxide,Morris, J. et al. (2019). "Biology: How Life Works", ...
ic bacteria and archaea fix carbon via the
reductive acetyl CoA pathway
Reduction, reduced, or reduce may refer to:
Science and technology Chemistry
* Reduction (chemistry), part of a reduction-oxidation (redox) reaction in which atoms have their oxidation state changed.
** Organic redox reaction, a redox reacti ...
, the
3-hydroxypropionate cycle
The 3-Hydroxypropionate bicycle, also known as the 3-Hydroxypropionate pathway, is a process that allows some bacteria to generate 3-Hydroxypropionic acid, 3-Hydroxypropionate utilizing carbon dioxide. In this pathway CO2 is fixed (i.e. incorporate ...
, or the
reverse Krebs cycle
The reverse Krebs cycle (also known as the reverse tricarboxylic acid cycle, the reverse TCA cycle, or the reverse citric acid cycle, or the reductive tricarboxylic acid cycle, or the reductive TCA cycle)
is a sequence of chemical reactions that ...
, these pathways are relatively small contributors to global carbon fixation compared to that catalyzed by RuBisCO.
Phosphoenolpyruvate carboxylase
Phosphoenolpyruvate carboxylase (also known as PEP carboxylase, PEPCase, or PEPC; , PDB ID: 3ZGE) is an enzyme in the family of carboxy-lyases found in plants and some bacteria that catalyzes the addition of bicarbonate (HCO3−) to phosphoenolp ...
, unlike RuBisCO, only temporarily fixes carbon. Reflecting its importance, RuBisCO is the most abundant protein in
leaves
A leaf (plural, : leaves) is any of the principal appendages of a vascular plant plant stem, stem, usually borne laterally aboveground and specialized for photosynthesis. Leaves are collectively called foliage, as in "autumn foliage", wh ...
, accounting for 50% of soluble leaf protein in
plants (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in
plants (5–9% of total leaf nitrogen).
Given its important role in the biosphere, the
genetic engineering
Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
of RuBisCO in crops is of continuing interest (see
below
Below may refer to:
*Earth
*Ground (disambiguation)
*Soil
*Floor
*Bottom (disambiguation)
Bottom may refer to:
Anatomy and sex
* Bottom (BDSM), the partner in a BDSM who takes the passive, receiving, or obedient role, to that of the top or ...
).
Structure
![RuBisCOActiveSite2](https://upload.wikimedia.org/wikipedia/commons/c/c5/RuBisCOActiveSite2.png)
In plants,
algae
Algae (; singular alga ) is an informal term for a large and diverse group of photosynthetic eukaryotic organisms. It is a polyphyletic grouping that includes species from multiple distinct clades. Included organisms range from unicellular mic ...
,
cyanobacteria
Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
, and
phototroph
Phototrophs () are organisms that carry out photon capture to produce complex organic compounds (e.g. carbohydrates) and acquire energy. They use the energy from light to carry out various cellular metabolic processes. It is a common misconcep ...
ic and
chemoautotrophic
A Chemotroph is an organism that obtains energy by the oxidation of electron donors in their environments. These molecules can be organic ( chemoorganotrophs) or inorganic (chemolithotrophs). The chemotroph designation is in contrast to phototro ...
Pseudomonadota
Pseudomonadota (synonym Proteobacteria) is a major phylum of Gram-negative bacteria. The renaming of phyla in 2021 remains controversial among microbiologists, many of whom continue to use the earlier names of long standing in the literature. The ...
(formerly proteobacteria), the enzyme usually consists of two types of protein subunit, called the large chain (L, about 55,000
Da) and the small chain (S, about 13,000 Da). The ''large-chain'' gene (''rbcL'') is encoded by the
chloroplast
A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it in ...
DNA in plants.
There are typically several related ''small-chain'' genes in the
nucleus
Nucleus ( : nuclei) is a Latin word for the seed inside a fruit. It most often refers to:
*Atomic nucleus, the very dense central region of an atom
*Cell nucleus, a central organelle of a eukaryotic cell, containing most of the cell's DNA
Nucle ...
of plant cells, and the small chains are imported to the
stromal compartment of chloroplasts from the
cytosol
The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells (intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...
by crossing the outer
chloroplast membrane
Chloroplasts contain several important membranes, vital for their function. Like mitochondria, chloroplasts have a double-membrane envelope, called the chloroplast envelope, but unlike mitochondria, chloroplasts also have internal membrane structu ...
.
['']Arabidopsis thaliana
''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land.
A winter a ...
'' has four RuBisCO small chain genes.
The enzymatically active
substrate (
ribulose
Ribulose is a ketopentose — a monosaccharide containing five carbon atoms, and including a ketone functional group. It has chemical formula . Two enantiomers are possible, -ribulose (-erythro-pentulose) and -ribulose (-erythro-pentulose). ...
1,5-bisphosphate)
binding site
In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may inclu ...
s are located in the large
chain
A chain is a serial assembly of connected pieces, called links, typically made of metal, with an overall character similar to that of a rope in that it is flexible and curved in compression but linear, rigid, and load-bearing in tension. A c ...
s that form
dimer
Dimer may refer to:
* Dimer (chemistry), a chemical structure formed from two similar sub-units
** Protein dimer, a protein quaternary structure
** d-dimer
* Dimer model, an item in statistical mechanics, based on ''domino tiling''
* Julius Dimer ...
s in which
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s from each large chain contribute to the binding sites. A total of eight large chains (= four dimers) and eight small chains assemble into a larger complex of about 540,000 Da. In some Pseudomonadota and
dinoflagellate
The dinoflagellates (Greek δῖνος ''dinos'' "whirling" and Latin ''flagellum'' "whip, scourge") are a monophyletic group of single-celled eukaryotes constituting the phylum Dinoflagellata and are usually considered algae. Dinoflagellates are ...
s, enzymes consisting of only large subunits have been found.
Magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
ion
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conve ...
s () are needed for enzymatic activity. Correct positioning of in the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate (binding site) a ...
of the enzyme involves addition of an "activating" carbon dioxide molecule (
) to a
lysine
Lysine (symbol Lys or K) is an α-amino acid that is a precursor to many proteins. It contains an α-amino group (which is in the protonated form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −C ...
in the active site (forming a
carbamate
In organic chemistry, a carbamate is a category of organic compounds with the general formula and structure , which are formally derived from carbamic acid (). The term includes organic compounds (e.g., the ester ethyl carbamate), formally o ...
). operates by driving deprotonation of the Lys210 residue, causing the Lys residue to rotate by 120 degrees to the ''trans'' conformer, decreasing the distance between the nitrogen of Lys and the carbon of . The close proximity allows for the formation of a covalent bond, resulting in the carbamate.
is first enabled to bind to the active site by the rotation of His335 to an alternate conformation. is then coordinated by the His residues of the active site (His300, His302, His335), and is partially neutralized by the coordination of three water molecules and their conversion to
−OH.
This coordination results in an unstable complex, but produces a favorable environment for the binding of . Formation of the carbamate is favored by an
alkaline
In chemistry, an alkali (; from ar, القلوي, al-qaly, lit=ashes of the saltwort) is a base (chemistry), basic, ionic compound, ionic salt (chemistry), salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as ...
pH. The pH and the
concentration
In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', ''number concentration'', an ...
of magnesium ions in the fluid compartment (in plants, the
stroma of the chloroplast) increases in the light. The role of changing pH and magnesium ion levels in the regulation of RuBisCO enzyme activity is discussed
below
Below may refer to:
*Earth
*Ground (disambiguation)
*Soil
*Floor
*Bottom (disambiguation)
Bottom may refer to:
Anatomy and sex
* Bottom (BDSM), the partner in a BDSM who takes the passive, receiving, or obedient role, to that of the top or ...
. Once the carbamate is formed, His335 finalizes the activation by returning to its initial position through thermal fluctuation.
Enzymatic activity
![RuBisCO reaction CO2 or O2](https://upload.wikimedia.org/wikipedia/commons/c/ce/RuBisCO_reaction_CO2_or_O2.svg)
RuBisCO is one of many enzymes in the
Calvin cycle
The Calvin cycle, light-independent reactions, bio synthetic phase, dark reactions, or photosynthetic carbon reduction (PCR) cycle of photosynthesis is a series of chemical reactions that convert carbon dioxide and hydrogen-carrier compounds into ...
. When Rubisco facilitates the attack of at the C2 carbon of RuBP and subsequent bond cleavage between the C3 and C2 carbon, 2 molecules of glycerate-3-phosphate are formed. The conversion involves these steps:
enolisation,
carboxylation
Carboxylation is a chemical reaction in which a carboxylic acid is produced by treating a substrate with carbon dioxide. The opposite reaction is decarboxylation. In chemistry, the term carbonation is sometimes used synonymously with carboxylatio ...
,
hydration Hydration may refer to:
* Hydrate, a substance that contains water
* Hydration enthalpy, energy released through hydrating a substance
* Hydration reaction, a chemical addition reaction where a hydroxyl group and proton are added to a compound
* ...
, C-C bond cleavage, and
protonation
In chemistry, protonation (or hydronation) is the adding of a proton (or hydron, or hydrogen cation), (H+) to an atom, molecule, or ion, forming a conjugate acid. (The complementary process, when a proton is removed from a Brønsted–Lowry acid, ...
.
Substrates
Substrates for RuBisCO are
ribulose-1,5-bisphosphate
Ribulose 1,5-bisphosphate (RuBP) is an organic substance that is involved in photosynthesis, notably as the principal acceptor in plants. It is a colourless anion, a double phosphate ester of the ketopentose (ketone-containing sugar with five car ...
and
carbon dioxide
Carbon dioxide (chemical formula ) is a chemical compound made up of molecules that each have one carbon atom covalently double bonded to two oxygen atoms. It is found in the gas state at room temperature. In the air, carbon dioxide is transpar ...
(distinct from the "activating" carbon dioxide). RuBisCO also catalyses a reaction of ribulose-1,5-bisphosphate and
molecular oxygen
There are several known allotropes of oxygen. The most familiar is molecular oxygen (O2), present at significant levels in Earth's atmosphere and also known as dioxygen or triplet oxygen. Another is the highly reactive ozone (O3). Others are:
*A ...
(O
2) instead of carbon dioxide ().
Discriminating between the substrates and O
2 is attributed to the differing interactions of the substrate's
quadrupole moment
A quadrupole or quadrapole is one of a sequence of configurations of things like electric charge or current, or gravitational mass that can exist in ideal form, but it is usually just part of a multipole expansion of a more complex structure ref ...
s and a high
electrostatic field
An electric field (sometimes E-field) is the physical field that surrounds electrically charged particles and exerts force on all other charged particles in the field, either attracting or repelling them. It also refers to the physical field f ...
gradient
In vector calculus, the gradient of a scalar-valued differentiable function of several variables is the vector field (or vector-valued function) \nabla f whose value at a point p is the "direction and rate of fastest increase". If the gradi ...
.
This gradient is established by the
dimer
Dimer may refer to:
* Dimer (chemistry), a chemical structure formed from two similar sub-units
** Protein dimer, a protein quaternary structure
** d-dimer
* Dimer model, an item in statistical mechanics, based on ''domino tiling''
* Julius Dimer ...
form of the minimally active RuBisCO, which with its two components provides a combination of oppositely charged domains required for the enzyme's interaction with O
2 and . These conditions help explain the low turnover rate found in RuBisCO: In order to increase the strength of the
electric field
An electric field (sometimes E-field) is the physical field that surrounds electrically charged particles and exerts force on all other charged particles in the field, either attracting or repelling them. It also refers to the physical field fo ...
necessary for sufficient interaction with the substrates’
quadrupole moment
A quadrupole or quadrapole is one of a sequence of configurations of things like electric charge or current, or gravitational mass that can exist in ideal form, but it is usually just part of a multipole expansion of a more complex structure ref ...
s, the C- and N- terminal segments of the enzyme must be closed off, allowing the active site to be isolated from the solvent and lowering the
dielectric constant
The relative permittivity (in older texts, dielectric constant) is the permittivity of a material expressed as a ratio with the electric permittivity of a vacuum. A dielectric is an insulating material, and the dielectric constant of an insulat ...
. This isolation has a significant
entropic
Entropy is a scientific concept, as well as a measurable physical property, that is most commonly associated with a state of disorder, randomness, or uncertainty. The term and the concept are used in diverse fields, from classical thermodynam ...
cost, and results in the poor turnover rate.
Binding RuBP
Carbamylation of the ε-amino group of Lys210 is stabilized by coordination with the . This reaction involves binding of the carboxylate termini of Asp203 and Glu204 to the ion. The substrate RuBP binds displacing two of the three aquo ligands.
Enolisation
Enolisation of RuBP is the conversion of the keto tautomer of RuBP to an enediol(ate).
Enolisation is initiated by deprotonation at C3. The enzyme base in this step has been debated,
but the steric constraints observed in crystal structures have made Lys210 the most likely candidate.
Specifically, the carbamate oxygen on Lys210 that is not coordinated with the Mg ion deprotonates the C3 carbon of RuBP to form a 2,3-enediolate.
Carboxylation
Carboxylation of the 2,3-enediolate results in the intermediate 3-keto-2′-carboxyarabinitol-1,5-bisphosphate and Lys334 is positioned to facilitate the addition of the substrate as it replaces the third -coordinated water molecule and add directly to the enediol. No Michaelis complex is formed in this process.
Hydration of this ketone results in an additional hydroxy group on C3, forming a gem-diol intermediate.
Carboxylation and hydration have been proposed as either a single concerted step
or as two sequential steps.
Concerted mechanism is supported by the proximity of the water molecule to C3 of RuBP in multiple crystal structures. Within the spinach structure, other residues are well placed to aid in the hydration step as they are within hydrogen bonding distance of the water molecule.
C-C bond cleavage
The gem-diol intermediate cleaves at the C2-C3 bond to form one molecule of glycerate-3-phosphate and a negatively charge carboxylate.
Stereo specific protonation of C2 of this carbanion results in another molecule of glycerate-3-phosphate. This step is thought to be facilitated by Lys175 or potentially the carbamylated Lys210.
Products
When carbon dioxide is the substrate, the product of the carboxylase reaction is an unstable six-carbon phosphorylated intermediate known as 3-keto-2-carboxyarabinitol-1,5-bisphosphate, which decays rapidly into two molecules of glycerate-3-phosphate. The 3-phosphoglycerate can be used to produce larger molecules such as
glucose
Glucose is a simple sugar with the molecular formula . Glucose is overall the most abundant monosaccharide, a subcategory of carbohydrates. Glucose is mainly made by plants and most algae during photosynthesis from water and carbon dioxide, using ...
.
When molecular oxygen is the substrate, the products of the oxygenase reaction are
phosphoglycolate and 3-phosphoglycerate. Phosphoglycolate is recycled through a sequence of reactions called
photorespiration
Photorespiration (also known as the oxidative photosynthetic carbon cycle or C2 cycle) refers to a process in plant metabolism where the enzyme RuBisCO oxygenates RuBP, wasting some of the energy produced by photosynthesis. The desired reaction i ...
, which involves enzymes and cytochromes located in the
mitochondria
A mitochondrion (; ) is an organelle found in the Cell (biology), cells of most Eukaryotes, such as animals, plants and Fungus, fungi. Mitochondria have a double lipid bilayer, membrane structure and use aerobic respiration to generate adenosi ...
and
peroxisomes
A peroxisome () is a membrane-bound organelle, a type of microbody, found in the cytoplasm of virtually all eukaryotic cells. Peroxisomes are oxidative organelles. Frequently, molecular oxygen serves as a co-substrate, from which hydrogen pero ...
(this is a case of
metabolite repair). In this process, two molecules of phosphoglycolate are converted to one molecule of carbon dioxide and one molecule of 3-phosphoglycerate, which can reenter the Calvin cycle. Some of the phosphoglycolate entering this pathway can be retained by plants to produce other molecules such as
glycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid (carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinogeni ...
. At ambient levels of carbon dioxide and oxygen, the ratio of the reactions is about 4 to 1, which results in a net carbon dioxide fixation of only 3.5. Thus, the inability of the enzyme to prevent the reaction with oxygen greatly reduces the photosynthetic capacity of many plants. Some plants, many algae, and photosynthetic bacteria have overcome this limitation by devising means to increase the concentration of carbon dioxide around the enzyme, including
carbon fixation,
crassulacean acid metabolism
Crassulacean acid metabolism, also known as CAM photosynthesis, is a carbon fixation pathway that evolved in some plants as an adaptation to arid conditions that allows a plant to photosynthesize during the day, but only exchange gases at night. ...
, and the use of
pyrenoid
Pyrenoids are sub-cellular micro-compartments found in chloroplasts of many algae,Giordano, M., Beardall, J., & Raven, J. A. (2005). CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Bio ...
.
Rubisco
side activities can lead to useless or inhibitory by-products. Important inhibitory by-products include
xylulose 1,5-bisphosphate
Xylulose is a ketopentose, a monosaccharide containing five carbon atoms, and including a ketone functional group. It has the chemical formula . In nature, it occurs in both the L- and D-enantiomers. 1-Deoxyxylulose is a precursor to terpenes vi ...
and
glycero-2,3-pentodiulose 1,5-bisphosphate, both caused by "misfires" halfway in the enolisation-carboxylation reaction. In higher plants, this process causes RuBisCO self-inhibition, which can be triggered by saturating and RuBP concentrations and solved by Rubisco activase.
Rate of enzymatic activity
Some enzymes can carry out thousands of chemical reactions each second. However, RuBisCO is slow, fixing only 3-10 carbon dioxide molecules each second per molecule of enzyme. The reaction catalyzed by RuBisCO is, thus, the primary rate-limiting factor of the Calvin cycle during the day. Nevertheless, under most conditions, and when light is not otherwise limiting photosynthesis, the speed of RuBisCO responds positively to increasing carbon dioxide concentration.
RuBisCO is usually only active during the day, as ribulose 1,5-bisphosphate is not regenerated in the dark. This is due to the regulation of several other enzymes in the Calvin cycle. In addition, the activity of RuBisCO is coordinated with that of the other enzymes of the Calvin cycle in several other ways:
By ions
Upon illumination of the chloroplasts, the
pH of the
stroma rises from 7.0 to 8.0 because of the proton (hydrogen ion, ) gradient created across the
thylakoid
Thylakoids are membrane-bound compartments inside chloroplasts and cyanobacteria. They are the site of the light-dependent reactions of photosynthesis. Thylakoids consist of a thylakoid membrane surrounding a thylakoid lumen. Chloroplast thyl ...
membrane. The movement of protons into thylakoids is
driven by light and is fundamental to
ATP synthesis
ATP synthase is a protein that catalyzes the formation of the energy storage molecule adenosine triphosphate (ATP) using adenosine diphosphate (ADP) and inorganic phosphate (Pi). It is classified under ligases as it changes ADP by the formation o ...
in chloroplasts ''(Further reading:
Photosynthetic reaction centre
A photosynthetic reaction center is a complex of several proteins, pigments and other co-factors that together execute the primary energy conversion reactions of photosynthesis. Molecular excitations, either originating directly from sunlight or t ...
;
Light-dependent reactions
Light-dependent reactions is jargon for certain photochemical reactions that are involved in photosynthesis, the main process by which plants acquire energy. There are two light dependent reactions, the first occurs at photosystem II (PSII) and ...
)''. To balance ion potential across the membrane, magnesium ions () move out of the thylakoids in response, increasing the concentration of magnesium in the stroma of the chloroplasts. RuBisCO has a high optimal pH (can be >9.0, depending on the magnesium ion concentration) and, thus, becomes "activated" by the introduction of carbon dioxide and magnesium to the active sites as described above.
By RuBisCO activase
In plants and some algae, another enzyme, RuBisCO activase (Rca, , ), is required to allow the rapid formation of the critical
carbamate
In organic chemistry, a carbamate is a category of organic compounds with the general formula and structure , which are formally derived from carbamic acid (). The term includes organic compounds (e.g., the ester ethyl carbamate), formally o ...
in the active site of RuBisCO.
This is required because
ribulose 1,5-bisphosphate
Ribulose 1,5-bisphosphate (RuBP) is an organic substance that is involved in photosynthesis, notably as the principal acceptor in plants. It is a colourless anion, a double phosphate ester of the ketopentose (ketone-containing sugar with five car ...
(RuBP) binds more strongly to the active sites of RuBisCO when excess carbamate is present, preventing processes form moving forward. In the light, RuBisCO activase promotes the release of the inhibitory (or — in some views — storage) RuBP from the catalytic sites of RuBisCO. Activase is also required in some plants (e.g., tobacco and many beans) because, in darkness, RuBisCO is inhibited (or protected from hydrolysis) by a competitive inhibitor synthesized by these plants, a substrate analog
2-Carboxy-D-arabitinol 1-phosphate
2-Carboxy-D-arabitinol 1-phosphate (or CA1P) is a molecule produced in plants that inhibits RuBisCO, a key enzyme in the Calvin cycle and carbon fixation. In dark conditions, this molecule binds to RuBisCO, preventing it from participating in che ...
(CA1P).
CA1P binds tightly to the active site of carbamylated RuBisCO and inhibits catalytic activity to an even greater extent. CA1P has also been shown to keep RuBisCO in a
conformation that is protected from
proteolysis
Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called protease ...
. In the light, RuBisCO activase also promotes the release of CA1P from the catalytic sites. After the CA1P is released from RuBisCO, it is rapidly converted to a non-inhibitory form by a light-activated
CA1P-phosphatase
The enzyme 2-carboxy-D-arabinitol-1-phosphatase (CA1Pase; EC 3.1.3.63) catalysis, catalyzes the reaction
:2-carboxy-D-arabinitol 1-phosphate + H2O \rightleftharpoons 2-carboxy-D-arabinitol + phosphate
This enzyme belongs to the family of hydrola ...
. Even without these strong inhibitors, once every several hundred reactions, the normal reactions with carbon dioxide or oxygen are not completed; other inhibitory substrate analogs are still formed in the active site. Once again, RuBisCO activase can promote the release of these analogs from the catalytic sites and maintain the enzyme in a catalytically active form. However, at high temperatures, RuBisCO activase aggregates and can no longer activate RuBisCO. This contributes to the decreased carboxylating capacity observed during heat stress.
By activase
The removal of the inhibitory RuBP, CA1P, and the other inhibitory substrate analogs by activase requires the consumption of
ATP. This reaction is inhibited by the presence of
ADP, and, thus, activase activity depends on the ratio of these compounds in the chloroplast stroma. Furthermore, in most plants, the sensitivity of activase to the ratio of ATP/ADP is modified by the stromal reduction/oxidation (
redox
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate (chemistry), substrate change. Oxidation is the loss of Electron, electrons or an increase in the oxidation state, while reduction ...
) state through another small regulatory protein,
thioredoxin
Thioredoxin is a class of small redox proteins known to be present in all organisms. It plays a role in many important biological processes, including redox signaling. In humans, thioredoxins are encoded by ''TXN'' and '' TXN2'' genes. Loss-of-fu ...
. In this manner, the activity of activase and the activation state of RuBisCO can be modulated in response to light intensity and, thus, the rate of formation of the ribulose 1,5-bisphosphate substrate.
By phosphate
In cyanobacteria, inorganic
phosphate
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phospho ...
(P
i) also participates in the co-ordinated regulation of photosynthesis: P
i binds to the RuBisCO active site and to another site on the large chain where it can influence transitions between activated and less active conformations of the enzyme. In this way, activation of bacterial RuBisCO might be particularly sensitive to P
i levels, which might cause it to act in a similar way to how RuBisCO activase functions in higher plants.
By carbon dioxide
Since carbon dioxide and oxygen
compete
Competition is a rivalry where two or more parties strive for a common goal which cannot be shared: where one's gain is the other's loss (an example of which is a zero-sum game). Competition can arise between entities such as organisms, indivi ...
at the active site of RuBisCO, carbon fixation by RuBisCO can be enhanced by increasing the carbon dioxide level in the compartment containing RuBisCO (
chloroplast stroma
A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant cell, plant and algae, algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, ...
). Several times during the evolution of plants, mechanisms have evolved for increasing the level of carbon dioxide in the stroma (see
carbon fixation). The use of oxygen as a substrate appears to be a puzzling process, since it seems to throw away captured energy. However, it may be a mechanism for preventing carbohydrate overload during periods of high light flux. This weakness in the enzyme is the cause of
photorespiration
Photorespiration (also known as the oxidative photosynthetic carbon cycle or C2 cycle) refers to a process in plant metabolism where the enzyme RuBisCO oxygenates RuBP, wasting some of the energy produced by photosynthesis. The desired reaction i ...
, such that healthy leaves in bright light may have zero net carbon fixation when the ratio of O
2 to available to RuBisCO shifts too far towards oxygen. This phenomenon is primarily temperature-dependent: High temperatures can decrease the concentration of dissolved in the moisture of leaf tissues. This phenomenon is also related to
water stress
Water scarcity (closely related to water stress or water crisis) is the lack of fresh water resources to meet the standard water demand. There are two types of water scarcity: physical or economic water scarcity. Physical water scarcity is where ...
: Since plant leaves are evaporatively cooled, limited water causes high leaf temperatures.
plants use the enzyme
PEP carboxylase initially, which has a higher affinity for . The process first makes a 4-carbon intermediate compound, which is shuttled into a site of
photosynthesis then de-carboxylated, releasing to boost the concentration of , hence the name plants.
Crassulacean acid metabolism
Crassulacean acid metabolism, also known as CAM photosynthesis, is a carbon fixation pathway that evolved in some plants as an adaptation to arid conditions that allows a plant to photosynthesize during the day, but only exchange gases at night. ...
(CAM) plants keep their
stoma
In botany, a stoma (from Greek ''στόμα'', "mouth", plural "stomata"), also called a stomate (plural "stomates"), is a pore found in the epidermis of leaves, stems, and other organs, that controls the rate of gas exchange. The pore is bor ...
ta closed during the day, which conserves water but prevents the light-independent reactions (a.k.a. the
Calvin Cycle
The Calvin cycle, light-independent reactions, bio synthetic phase, dark reactions, or photosynthetic carbon reduction (PCR) cycle of photosynthesis is a series of chemical reactions that convert carbon dioxide and hydrogen-carrier compounds into ...
) from taking place, since these reactions require to pass by gas exchange through these openings. Evaporation through the upper side of a leaf is prevented by a layer of
wax
Waxes are a diverse class of organic compounds that are lipophilic, malleable solids near ambient temperatures. They include higher alkanes and lipids, typically with melting points above about 40 °C (104 °F), melting to giv ...
.
Genetic engineering
Since RuBisCO is often rate-limiting for photosynthesis in plants, it may be possible to improve
photosynthetic efficiency The photosynthetic efficiency is the fraction of light energy converted into chemical energy during photosynthesis in green plants and algae. Photosynthesis can be described by the simplified chemical reaction
:6 H2O + 6 CO2 + energy → C6H12O6 + ...
by modifying RuBisCO genes in plants to increase catalytic activity and/or decrease oxygenation rates.
This could improve
biosequestration
Biosequestration or biological sequestration is the capture and storage of the atmospheric greenhouse gas carbon dioxide by continual or enhanced biological processes.
This form of carbon sequestration occurs through increased rates of photosyn ...
of and be both an important
climate change
In common usage, climate change describes global warming—the ongoing increase in global average temperature—and its effects on Earth's climate system. Climate change in a broader sense also includes previous long-term changes to E ...
strategy and a strategy to increase crop yields.
Approaches under investigation include transferring RuBisCO genes from one organism into another organism, engineering Rubisco activase from thermophilic cyanobacteria into temperature sensitive plants, increasing the level of expression of RuBisCO subunits, expressing RuBisCO small chains from the
chloroplast DNA
Chloroplast DNA (cpDNA) is the DNA located in chloroplasts, which are photosynthetic organelles located within the cells of some eukaryotic organisms. Chloroplasts, like other types of plastid, contain a genome separate from that in the cell nu ...
, and altering RuBisCO genes to increase specificity for carbon dioxide or otherwise increase the rate of carbon fixation.
Mutagenesis in plants
In general,
site-directed mutagenesis
Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesi ...
of RuBisCO has been mostly unsuccessful,
though mutated forms of the protein have been achieved in tobacco plants with subunit C
4 species, and a RuBisCO with more C
4-like kinetic characteristics have been attained in rice via nuclear transformation. Robust and reliable engineering for yield of RuBisCO and other enzymes in the C
3 cycle was shown to be possible, and it was first achieved in 2019 through a synthetic biology approach.
One avenue is to introduce RuBisCO variants with naturally high specificity values such as the ones from the
red alga
Red algae, or Rhodophyta (, ; ), are one of the oldest groups of eukaryotic algae. The Rhodophyta also comprises one of the largest phyla of algae, containing over 7,000 currently recognized species with taxonomic revisions ongoing. The majority ...
''Galdieria partita'' into plants. This may improve the photosynthetic efficiency of crop plants, although possible negative impacts have yet to be studied. Advances in this area include the replacement of the tobacco enzyme with that of the purple photosynthetic bacterium ''
Rhodospirillum rubrum
''Rhodospirillum rubrum'' (''R. rubrum'') is a Gram-negative, pink-coloured bacterium, with a size of 800 to 1000 nanometers. It is a facultative anaerobe, thus capable of using oxygen for aerobic respiration under aerobic conditions, or an alte ...
''. In 2014, two transplastomic tobacco lines with functional RuBisCO from the
cyanobacterium
Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blue ...
''
Synechococcus
''Synechococcus'' (from the Greek ''synechos'', in succession, and the Greek ''kokkos'', granule) is a unicellular cyanobacterium that is very widespread in the marine environment. Its size varies from 0.8 to 1.5 µm. The photosynthetic c ...
elongatus'' PCC7942 (Se7942) were created by replacing the RuBisCO with the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35. Both mutants had increased fixation rates when measured as carbon molecules per RuBisCO. However, the mutant plants grew more slowly than wild-type.
A recent theory explores the trade-off between the relative specificity (i.e., ability to favour fixation over O
2 incorporation, which leads to the energy-wasteful process of
photorespiration
Photorespiration (also known as the oxidative photosynthetic carbon cycle or C2 cycle) refers to a process in plant metabolism where the enzyme RuBisCO oxygenates RuBP, wasting some of the energy produced by photosynthesis. The desired reaction i ...
) and the rate at which product is formed. The authors conclude that RuBisCO may actually have evolved to reach a point of 'near-perfection' in many plants (with widely varying substrate availabilities and environmental conditions), reaching a compromise between specificity and reaction rate.
It has been also suggested that the oxygenase reaction of RuBisCO prevents depletion near its active sites and provides the maintenance of the chloroplast redox state.
Since photosynthesis is the single most effective natural regulator of
carbon dioxide in the Earth's atmosphere
Carbon dioxide in Earth's atmosphere is a trace gas that plays an integral part in the greenhouse effect, carbon cycle, photosynthesis and oceanic carbon cycle. It is one of several greenhouse gases in Earth's atmosphere that are contributing t ...
, a biochemical model of RuBisCO reaction is used as the core module of climate change models. Thus, a correct model of this reaction is essential to the basic understanding of the relations and interactions of environmental models.
Expression in bacterial hosts
There currently are very few effective methods for expressing functional plant Rubisco in bacterial hosts for genetic manipulation studies. This is largely due to Rubisco's requirement of complex cellular machinery for its biogenesis and metabolic maintenance including the nuclear-encoded RbcS subunits, which are typically imported into
chloroplast
A chloroplast () is a type of membrane-bound organelle known as a plastid that conducts photosynthesis mostly in plant and algal cells. The photosynthetic pigment chlorophyll captures the energy from sunlight, converts it, and stores it in ...
s as unfolded proteins. Furthermore, sufficient expression and interaction with Rubisco activase are major challenges as well.
One successful method for expression of Rubisco in
E. coli involves the co-expression of multiple chloroplast chaperones, though this has only been shown for ''
Arabidopsis thaliana
''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small flowering plant native to Eurasia and Africa. ''A. thaliana'' is considered a weed; it is found along the shoulders of roads and in disturbed land.
A winter a ...
'' Rubisco.
Depletion in proteomic studies
Due to its high abundance in plants (generally 40% of the total protein content), RuBisCO often impedes analysis of important signaling proteins such as
transcription factor
In molecular biology, a transcription factor (TF) (or sequence-specific DNA-binding factor) is a protein that controls the rate of transcription of genetic information from DNA to messenger RNA, by binding to a specific DNA sequence. The fu ...
s,
kinase
In biochemistry, a kinase () is an enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the high-energy ATP molecule don ...
s, and regulatory proteins found in lower abundance (10-100 molecules per cell) within plants.
For example, using
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
on plant protein mixtures would result in multiple intense RuBisCO subunit peaks that interfere and hide those of other proteins.
Recently, one efficient method for precipitating out RuBisCO involves the usage of
protamine sulfate
Protamine sulfate is a medication that is used to reverse the effects of heparin. It is specifically used in heparin overdose, in low molecular weight heparin overdose, and to reverse the effects of heparin during delivery and heart surgery. ...
solution. Other existing methods for depleting RuBisCO and studying lower abundance proteins include
fractionation
Fractionation is a separation process in which a certain quantity of a mixture (of gases, solids, liquids, enzymes, or isotopes, or a suspension) is divided during a phase transition, into a number of smaller quantities (fractions) in which the ...
techniques with calcium and phytate,
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
with polyethylene glycol,
affinity chromatography
Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...
, and aggregation using
DTT, though these methods are more time-consuming and less efficient when compared to protamine sulfate precipitation.
Evolution of RuBisCO
Phylogenetic studies
The chloroplast gene ''rbcL'', which codes for the large subunit of RuBisCO has been widely used as an appropriate
locus
Locus (plural loci) is Latin for "place". It may refer to:
Entertainment
* Locus (comics), a Marvel Comics mutant villainess, a member of the Mutant Liberation Front
* ''Locus'' (magazine), science fiction and fantasy magazine
** ''Locus Award' ...
for analysis of
phylogenetics
In biology, phylogenetics (; from Greek language, Greek wikt:φυλή, φυλή/wikt:φῦλον, φῦλον [] "tribe, clan, race", and wikt:γενετικός, γενετικός [] "origin, source, birth") is the study of the evolutionary his ...
in plant taxonomy.
Origin
Non-carbon-fixing proteins similar to RuBisCO, termed RuBisCO-like proteins (RLPs), are also found in the wild in organisms as common as ''
Bacillus subtilis
''Bacillus subtilis'', known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacillu ...
''. This bacterium has a rbcL-like protein with a
2,3-diketo-5-methylthiopentyl-1-phosphate enolase
2,3-diketo-5-methylthiopentyl-1-phosphate enolase (, ''DK-MTP-1-P enolase'', ''MtnW'', ''YkrW'', ''RuBisCO-like protein'', ''RLP'') is an enzyme with List of enzymes, systematic name ''2,3-diketo-5-methylthiopentyl-1-phosphate keto-enol-isomerase' ...
function, part of the
methionine salvage pathway
Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical rol ...
. Later identifications found functionally divergent examples dispersed all over bacteria and archaea, as well as transitionary enzymes performing both RLP-type enolase and RuBisCO functions. It is now believed that the current RuBisCO evolved from a dimeric RLP ancestor, acquiring its carboxylase function first before further oligomerizing and then recruiting the small subunit to form the familiar modern enzyme.
The small subunit probably first evolved in anaerobic and thermophilic organisms, where it enabled RuBisCO to catalyze its reaction at higher temperatures. In addition to its effect on stabilizing catalysis, it enabled the evolution of higher specificities for over O
2 by modulating the effect that substitutions within RuBisCO have on enzymatic function. Substitutions that do not have an effect without the small subunit suddenly become beneficial when it is bound. Furthermore, the small subunit enabled the accumulation of substitutions that are only tolerated in its presence. Accumulation of such substitutions leads to a strict dependence on the small subunit, which is observed in extant Rubiscos that bind a small subunit.
C4
With the mass convergent evolution of the
C4-fixation pathway in a diversity of plant lineages, ancestral C
3-type RuBisCO evolved to have faster turnover of in exchange for lower specificity as a result of the greater localization of from the
mesophyll cell
A leaf (plural, : leaves) is any of the principal appendages of a vascular plant plant stem, stem, usually borne laterally aboveground and specialized for photosynthesis. Leaves are collectively called foliage, as in "autumn foliage", wh ...
s into the
bundle sheath cells. This was achieved through enhancement of conformational flexibility of the “open-closed” transition in the
Calvin Cycle
The Calvin cycle, light-independent reactions, bio synthetic phase, dark reactions, or photosynthetic carbon reduction (PCR) cycle of photosynthesis is a series of chemical reactions that convert carbon dioxide and hydrogen-carrier compounds into ...
. Laboratory-based phylogenetic studies have shown that this evolution was constrained by the trade-off between stability and activity brought about by the series of necessary
mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
s for C
4 RuBisCO.
Moreover, in order to sustain the destabilizing mutations, the evolution to C
4 RuBisCO was preceded by a period in which mutations granted the enzyme increased stability, establishing a buffer to sustain and maintain the mutations required for C
4 RuBisCO. To assist with this buffering process, the newly-evolved enzyme was found to have further developed a series of stabilizing mutations. While RuBisCO has always been accumulating new mutations, most of these mutations that have survived have not had significant effects on protein stability. The destabilizing C
4 mutations on RuBisCO has been sustained by environmental pressures such as low concentrations, requiring a sacrifice of stability for new adaptive functions.
History of the term
The term "RuBisCO" was coined humorously in 1979, by
David Eisenberg
David S. Eisenberg (born 15 March 1939) is an American biochemist and biophysicist best known for his contributions to structural biology and computational molecular biology, a professor at the University of California, Los Angeles since the earl ...
at a seminar honouring the retirement of the early, prominent RuBisCO researcher,
Sam Wildman
Samuel Goodnow Wildman (May 26, 1912 – August 16, 2004) was an American biologist. Wildman joined the University of California, Los Angeles, as a professor of biology in 1950 and retired in 1979. Professor Wildman is best known for his leading ...
, and also alluded to the snack food trade name "
Nabisco
Nabisco (, abbreviated from the earlier name National Biscuit Company) is an American manufacturer of cookies and snacks headquartered in East Hanover, New Jersey. The company is a subsidiary of Illinois-based Mondelēz International.
Nabisco's ...
" in reference to Wildman's attempts to create an edible protein supplement from tobacco leaves.
The capitalization of the name has been long debated. It can be capitalized for each letter of the full name (Ribulose-1,5 bisphosphate carboxylase/oxygenase), but it has also been argued that is should all be in lower case (rubisco), similar to other terms like scuba or laser.
See also
*
Carbon cycle
The carbon cycle is the biogeochemical cycle by which carbon is exchanged among the biosphere, pedosphere, geosphere, hydrosphere, and Earth's atmosphere, atmosphere of the Earth. Carbon is the main component of biological compounds as well as ...
*
Photorespiration
Photorespiration (also known as the oxidative photosynthetic carbon cycle or C2 cycle) refers to a process in plant metabolism where the enzyme RuBisCO oxygenates RuBP, wasting some of the energy produced by photosynthesis. The desired reaction i ...
*
Pyrenoid
Pyrenoids are sub-cellular micro-compartments found in chloroplasts of many algae,Giordano, M., Beardall, J., & Raven, J. A. (2005). CO2 concentrating mechanisms in algae: mechanisms, environmental modulation, and evolution. Annu. Rev. Plant Bio ...
*
C3 carbon fixation
carbon fixation is the most common of three metabolic pathways for carbon fixation in photosynthesis, along with and CAM. This process converts carbon dioxide and ribulose bisphosphate (RuBP, a 5-carbon sugar) into two molecules of 3-phosph ...
*
C4 carbon fixation
carbon fixation or the Hatch–Slack pathway is one of three known photosynthetic processes of carbon fixation in plants. It owes the names to the 1960's discovery by Marshall Davidson Hatch and Charles Roger Slack that some plants, when sup ...
*
Crassulacean acid metabolism
Crassulacean acid metabolism, also known as CAM photosynthesis, is a carbon fixation pathway that evolved in some plants as an adaptation to arid conditions that allows a plant to photosynthesize during the day, but only exchange gases at night. ...
/CAM photosynthesis
*
Carboxysome
Carboxysomes are bacterial microcompartments (BMCs) consisting of polyhedral protein shells filled with the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)—the predominant enzyme in carbon fixation and the rate limiting e ...
References
Further reading
*
*
External links
*
{{Enzymes
Photosynthesis
EC 4.1.1