View source for RuBisCO

{{short description|Key enzyme of photosynthesis involved in carbon fixation}}
{{Infobox enzyme
|name=Ribulose-1,5-bisphosphate carboxylase oxygenase| image = SpinachRuBisCO.png
| caption =A 3d depiction of the activated RuBisCO from spinach in open form with active site accessible. The active site Lys175 residues are marked in pink, and a close-up of the residue is provided to the right for one of the monomers composing the enzyme.
| EC_number = 4.1.1.39
| CAS_number = 9027-23-0
| GO_code = 0016984
| Name = Ribulose-1,5-biphosphate carboxylase/oxygenase
| width = 
}}

'''Ribulose-1,5-bisphosphate carboxylase/oxygenase''', commonly known by the abbreviations '''RuBisCo''', '''rubisco''',<ref name="Sharkey_2018">{{cite journal | vauthors = Sharkey TD | author-link = Thomas D. Sharkey | title = Discovery of the canonical Calvin-Benson cycle | journal = Photosynthesis Research | volume = 140 | issue = 2 | pages = 235–252 | date = May 2019 | pmid = 30374727 | doi = 10.1007/s11120-018-0600-2 | bibcode = 2019PhoRe.140..235S | osti = 1607740 | s2cid = 53092349 }}</ref> '''RuBPCase''',<ref>{{Cite journal |last1=Nivison |first1=Helen |first2=C. |last2=Stocking |title=Ribulose Bisphosphate Carboxylase Synthesis in Barley Leaves |journal=Plant Physiology|year=1983 |volume=73 |issue=4 |pages=906–911 |doi=10.1104/pp.73.4.906 |pmid=16663341 |pmc=1066578 }}</ref> or '''RuBPco''',<ref>{{cite journal |last1=Mächler |first1=Felix |first2=Josef |last2= Nösberger |title=Bicarbonate Inhibits Ribulose-1,5-Bisphosphate Carboxylase |journal=Plant Physiology|year=1988 |volume=88 |issue=2 |pages=462–465 |doi=10.1104/pp.88.2.462 |pmid=16666327 |pmc=1055600 }}</ref> is an [[enzyme]] ({{EnzExplorer|4.1.1.39}}) involved in the [[Photosynthesis#Light-independent reactions|light-independent (or "dark") part of photosynthesis]], including the [[carbon fixation]] by which atmospheric [[carbon dioxide]] is converted by plants and other [[photosynthesis|photosynthetic]] organisms to [[fuel|energy-rich]] [[molecule]]s such as [[glucose]]. It emerged approximately four billion years ago in primordial metabolism prior to the presence of oxygen on Earth.<ref>[https://www.mpg.de/19348003/1010-terr-back-to-the-future-of-photosynthesis-153410-x Back to the future of photosynthesis: Resurrecting billon-year-old enzymes reveals how photosynthesis adapted to the rise of oxygen.], News from the Max Planck Society, October 13, 2022</ref>  It is probably the most abundant enzyme on Earth. In chemical terms, it [[catalysis|catalyzes]] the [[carboxylation]] of [[ribulose-1,5-bisphosphate]] (also known as RuBP).<ref>{{cite book | vauthors = Cooper GM |title=The Cell: A Molecular Approach |publisher=ASM Press |location=Washington, D.C. |year=2000 |isbn=978-0-87893-106-4 |edition=2nd |chapter=10.The Chloroplast Genome |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK9905/ |quote=, one of the subunits of ribulose bisphosphate carboxylase (rubisco) is encoded by chloroplast DNA. Rubisco is the critical enzyme that catalyzes the addition of {{CO2}} to ribulose-1,5-bisphosphate during the Calvin cycle. It is also thought to be the single most abundant protein on Earth, so it is noteworthy that one of its subunits is encoded by the chloroplast genome. |url-access=registration |url=https://archive.org/details/cell00geof }}</ref><ref name = "Dhingra_2004">{{cite journal | vauthors = Dhingra A, Portis AR, Daniell H | title = Enhanced translation of a chloroplast-expressed RbcS gene restores small subunit levels and photosynthesis in nuclear RbcS antisense plants | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 101 | issue = 16 | pages = 6315–6320 | date = April 2004 | pmid = 15067115 | pmc = 395966 | doi = 10.1073/pnas.0400981101 | quote = (Rubisco) is the most prevalent enzyme on this planet, accounting for 30–50% of total soluble protein in the chloroplast; | doi-access = free | bibcode = 2004PNAS..101.6315D }}</ref><ref name="Feller_2008">{{cite journal | vauthors = Feller U, Anders I, Mae T | title = Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated | journal = Journal of Experimental Botany | volume = 59 | issue = 7 | pages = 1615–1624 | year = 2008 | pmid = 17975207 | doi = 10.1093/jxb/erm242 | doi-access = free }}</ref>

==Alternative carbon fixation pathways==
RuBisCO is important [[biology|biologically]] because it catalyzes the primary [[chemical reaction]] by which [[Total inorganic carbon|inorganic carbon]] enters the [[biosphere]]. While many [[autotroph]]ic bacteria and archaea fix carbon via the [[reductive acetyl CoA Pathway|reductive acetyl CoA pathway]], the [[3-hydroxypropionate cycle]], or the [[reverse Krebs cycle]], these pathways are relatively small contributors to global carbon fixation compared to that catalyzed by RuBisCO. [[Phosphoenolpyruvate carboxylase]], unlike RuBisCO, only temporarily fixes carbon. Reflecting its importance, RuBisCO is the most abundant protein in [[leaf|leaves]], accounting for 50% of soluble leaf protein in [[C3 carbon fixation|{{C3}} plants]] (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in [[C4 carbon fixation|{{C4}} plants]] (5–9% of total leaf nitrogen).<ref name="Feller_2008"/> Given its important role in the biosphere, the [[genetic engineering]] of RuBisCO in crops is of continuing interest (see [[#Genetic engineering|below]]).

== Structure ==
[[File:RuBisCOActiveSite2.png|thumb|Active site of RuBisCO of ''[[Galdieria sulphuraria]]'' with {{CO2}}: Residues involved in both the active site and stabilizing {{CO2}} for enzyme catalysis are shown in color and labeled. Distances of the hydrogen bonding interactions are shown in angstroms. {{chem2|Mg(2+)}} ion (green sphere) is shown coordinated to {{CO2}}, and is followed by three water molecules (red spheres). All other residues are placed in grayscale.]]
[[File:Plastomap of Arabidopsis thaliana.svg|thumb|Location of the ''rbcL'' gene in the [[chloroplast genome]] of ''[[Arabidopsis thaliana]]'' (positions ca. 55-56.4 kb). ''rbcL'' is one of the 21 protein-coding genes involved in photosynthesis (green boxes).]]
In plants, [[algae]], [[cyanobacteria]], and [[phototroph]]ic and [[Chemotroph|chemoautotrophic]] [[Pseudomonadota]] (formerly referred to as proteobacteria), the enzyme usually consists of two types of protein subunit, called the large chain ('''L''', about 55,000 [[Atomic mass unit|Da]]) and the small chain ('''S''', about 13,000 Da). The ''large-chain'' gene (''rbcL'') is encoded by the [[chloroplast]] DNA in plants.<ref name="pmid29594130">{{cite journal | vauthors = Vitlin Gruber A, Feiz L | title = Rubisco Assembly in the Chloroplast | journal = Frontiers in Molecular Biosciences | volume = 5 | issue = | article-number = 24 | date = 2018 | pmid = 29594130 | pmc = 5859369 | doi = 10.3389/fmolb.2018.00024 | doi-access = free }}</ref> There are typically several related ''small-chain'' genes in the [[Cell nucleus|nucleus]] of plant cells, and the small chains are imported to the [[stromal]] compartment of chloroplasts from the [[cytosol]] by crossing the outer [[chloroplast membrane]].<ref name = "Dhingra_2004" /><ref name="pmid11401297">''[[Arabidopsis thaliana]]'' has four RuBisCO small chain genes.<br /> {{cite journal | vauthors = Yoon M, Putterill JJ, Ross GS, Laing WA | title = Determination of the relative expression levels of rubisco small subunit genes in Arabidopsis by rapid amplification of cDNA ends | journal = Analytical Biochemistry | volume = 291 | issue = 2 | pages = 237–244 | date = April 2001 | pmid = 11401297 | doi = 10.1006/abio.2001.5042 }}</ref> The enzymatically active [[substrate (biochemistry)|substrate]] ([[ribulose]] 1,5-bisphosphate) [[active site|binding site]]s are located in the large [[polymer|chain]]s that form [[protein dimer|dimer]]s in which [[amino acid]]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.<ref>{{cite book | vauthors = Stryer L, Berg JM, Tymoczko JL |title=Biochemistry |publisher=W.H. Freeman |location=San Francisco |year=2002 |isbn=978-0-7167-3051-4 |edition=5th |chapter= Chapter 20: The Calvin Cycle and the Pentose Phosphate Pathway |quote= Figure 20.3.  Structure of Rubisco.] (Color-coded ribbon diagram) |chapter-url=https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer |url-access=registration |url=https://archive.org/details/biochemistrychap00jere }}<br /></ref> In some Pseudomonadota and [[dinoflagellate]]s, enzymes consisting of only large subunits have been found.{{efn|name=comp|1=The structure of RuBisCO from the photosynthetic bacterium ''[[Rhodospirillaceae|Rhodospirillum rubrum]]'' has been determined by [[X-ray crystallography]], see: {{Protein Data Bank|9RUB}}. A comparison of the structures of [[eukaryotic]] and [[bacterial]] RuBisCO is shown in the [[Protein Data Bank]] "Molecule of the Month" #11.<ref>{{cite journal | date = November 2000 | vauthors = Goodsell D | title = Rubisco | url = https://pdb101.rcsb.org/motm/11 | journal = Molecule of the Month | publisher = RCSB PDB (Research Collaboratory for Structural Bioinformatics PDB) | doi = 10.2210/rcsb_pdb/mom_2000_11| url-access = subscription }}</ref>}}

[[Magnesium]] [[ion]]s ({{chem2|Mg(2+)}}) are needed for enzymatic activity. Correct positioning of {{chem2|Mg(2+)}} in the [[active site]] of the enzyme involves addition of an "activating" carbon dioxide molecule ([[Carbon dioxide|{{CO2}}]]) to a [[lysine]] in the active site (forming a [[carbamate]]).<ref>{{cite web | url = https://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.4496 | title = Molecular Cell Biology | edition = 4th | vauthors = Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell JE | publisher = W. H. Freeman & Co. | date = 2000 | location = New York }}{{dead link|date=July 2025|bot=medic}}{{cbignore|bot=medic}} Figure 16-48 shows a structural model of the active site, including the involvement of magnesium.</ref> {{chem2|Mg(2+)}} 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 {{CO2|link=yes}}. The close proximity allows for the formation of a covalent bond, resulting in the carbamate.<ref name="Stec_2012">{{cite journal | vauthors = Stec B | title = Structural mechanism of RuBisCO activation by carbamylation of the active site lysine | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 46 | pages = 18785–18790 | date = November 2012 | pmid = 23112176 | pmc = 3503183 | doi = 10.1073/pnas.1210754109 | doi-access = free | bibcode = 2012PNAS..10918785S }}</ref> {{chem2|Mg(2+)}} is first enabled to bind to the active site by the rotation of His335 to an alternate conformation. {{chem2|Mg(2+)}} 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 <sup>−</sup>OH.<ref name="Stec_2012" /> This coordination results in an unstable complex, but produces a favorable environment for the binding of {{chem2|Mg(2+)}}. Formation of the carbamate is favored by an [[alkalinity|alkaline]] [[pH]]. The pH and the [[concentration]] of magnesium ions in the fluid compartment (in plants, the [[Chloroplast|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 [[#By ions|below]]. Once the carbamate is formed, His335 finalizes the activation by returning to its initial position through thermal fluctuation.<ref name="Stec_2012" />

{|
|- valign=top
|{{Infobox protein family
| Symbol = RuBisCO_large
| Name = RuBisCO large chain,<br />catalytic domain
| image = 
| width = 
| caption = 
| Pfam = PF00016
| InterPro = IPR000685
| SMART = 
| PROSITE = PDOC00142
| SCOP = 3rub
| TCDB = 
| OPM family = 
| OPM protein = 
| CDD = cd08148
| PDB = {{PDB2|1aa1}}, {{PDB2|1aus}}, {{PDB2|1bwv}}, {{PDB2|1bxn}}, {{PDB2|1ej7}}, {{PDB2|1geh}}, {{PDB2|1gk8}}, {{PDB2|1ir1}}, {{PDB2|1ir2}}, {{PDB2|1iwa}}, {{PDB2|1rba}}, {{PDB2|1rbl}}, {{PDB2|1rbo}}, {{PDB2|1rco}}, {{PDB2|1rcx}}, {{PDB2|1rld}}, {{PDB2|1rsc}}, {{PDB2|1rus}}, {{PDB2|1rxo}}, {{PDB2|1svd}}, {{PDB2|1tel}}, {{PDB2|1upm}}, {{PDB2|1upp}}, {{PDB2|1uw9}}, {{PDB2|1uwa}}, {{PDB2|1uzd}}, {{PDB2|1uzh}}, {{PDB2|1wdd}}, {{PDB2|1ykw}}, {{PDB2|2cwx}}, {{PDB2|2cxe}}, {{PDB2|2d69}}, {{PDB2|2qyg}}, {{PDB2|2rus}}, {{PDB2|2v63}}, {{PDB2|2v67}}, {{PDB2|2v68}}, {{PDB2|2v69}}, {{PDB2|2v6a}}, {{PDB2|3rub}}, {{PDB2|4rub}}, {{PDB2|5rub}}, {{PDB2|8ruc}}, {{PDB2|9rub}}
}}
|{{Infobox protein family
| Symbol = RuBisCO_large_N
| Name = RuBisCO, N-terminal domain
| image = 
| width = 
| caption = 
| Pfam = PF02788
| InterPro = IPR017444
| SMART = 
| PROSITE = 
| SCOP = 3rub
| TCDB = 
| OPM family = 
| OPM protein = 
| PDB = {{PDB2|1aa1}}, {{PDB2|1aus}}, {{PDB2|1bwv}}, {{PDB2|1bxn}}, {{PDB2|1ej7}}, {{PDB2|1geh}}, {{PDB2|1gk8}}, {{PDB2|1ir1}}, {{PDB2|1ir2}}, {{PDB2|1iwa}}, {{PDB2|1rba}}, {{PDB2|1rbl}}, {{PDB2|1rbo}}, {{PDB2|1rco}}, {{PDB2|1rcx}}, {{PDB2|1rld}}, {{PDB2|1rsc}}, {{PDB2|1rus}}, {{PDB2|1rxo}}, {{PDB2|1svd}}, {{PDB2|1tel}}, {{PDB2|1upm}}, {{PDB2|1upp}}, {{PDB2|1uw9}}, {{PDB2|1uwa}}, {{PDB2|1uzd}}, {{PDB2|1uzh}}, {{PDB2|1wdd}}, {{PDB2|1ykw}}, {{PDB2|2cwx}}, {{PDB2|2cxe}}, {{PDB2|2d69}}, {{PDB2|2qyg}}, {{PDB2|2rus}}, {{PDB2|2v63}}, {{PDB2|2v67}}, {{PDB2|2v68}}, {{PDB2|2v69}}, {{PDB2|2v6a}}, {{PDB2|3rub}}, {{PDB2|4rub}}, {{PDB2|5rub}}, {{PDB2|8ruc}}, {{PDB2|9rub}}
}}
|{{Infobox protein family
| Symbol = RuBisCO_small
| Name = RuBisCO, small chain
| image = 
| width = 
| caption = 
| Pfam = PF00101
| InterPro = IPR000894
| SMART = 
| PROSITE = 
| SCOP = 3rub
| TCDB = 
| OPM family = 
| OPM protein = 
| CDD = cd03527
| PDB = {{PDB2|1aa1}}, {{PDB2|1aus}}, {{PDB2|1bwv}}, {{PDB2|1bxn}}, {{PDB2|1ej7}}, {{PDB2|1gk8}}, {{PDB2|1ir1}}, {{PDB2|1ir2}}, {{PDB2|1iwa}}, {{PDB2|1rbl}}, {{PDB2|1rbo}}, {{PDB2|1rco}}, {{PDB2|1rcx}}, {{PDB2|1rlc}}, {{PDB2|1rld}}, {{PDB2|1rsc}}, {{PDB2|1rxo}}, {{PDB2|1svd}}, {{PDB2|1upm}}, {{PDB2|1upp}}, {{PDB2|1uw9}}, {{PDB2|1uwa}}, {{PDB2|1uzd}}, {{PDB2|1uzh}}, {{PDB2|1wdd}}, {{PDB2|2v63}}, {{PDB2|2v67}}, {{PDB2|2v68}}, {{PDB2|2v69}}, {{PDB2|2v6a}}, {{PDB2|3rub}}, {{PDB2|4rub}}, {{PDB2|8ruc}}
}}
|}

==Enzymatic activity==
[[File:RuBisCO reaction CO2 or O2.svg|center|thumb|upright=2|Two main reactions of RuBisCo: {{CO2}} fixation and oxygenation.]]
RuBisCO is one of many enzymes in the [[Calvin cycle]]. When RuBisCO facilitates the attack of {{CO2}} 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: [[Keto-enol tautomerism|enolisation]], [[carboxylation]], [[Hydration reaction|hydration]], C-C bond cleavage, and [[protonation]].<ref name="Andersson_2008">{{cite journal | vauthors = Andersson I | title = Catalysis and regulation in Rubisco | journal = Journal of Experimental Botany | volume = 59 | issue = 7 | pages = 1555–1568 | date = May 2008 | pmid = 18417482 | doi = 10.1093/jxb/ern091 | doi-access = free }}</ref><ref name = "Erb_2018">{{cite journal | vauthors = Erb TJ, Zarzycki J | title = A short history of RubisCO: the rise and fall (?) of Nature's predominant {{CO2}} fixing enzyme | journal = Current Opinion in Biotechnology | volume = 49 | pages = 100–107 | date = February 2018 | pmid = 28843191 | pmc = 7610757 | doi = 10.1016/j.copbio.2017.07.017 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Lundqvist T, Schneider G | title = Crystal structure of activated ribulose-1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5-bisphosphate | journal = The Journal of Biological Chemistry | volume = 266 | issue = 19 | pages = 12604–12611 | date = July 1991 | pmid = 1905726 | doi = 10.1016/S0021-9258(18)98942-8 | doi-access = free }}</ref>

=== Substrates ===
[[Substrate (chemistry)|Substrate]]s for RuBisCO are [[ribulose-1,5-bisphosphate]] and [[carbon dioxide]] (distinct from the "activating" carbon dioxide). RuBisCO also catalyses a reaction of ribulose-1,5-bisphosphate and [[oxygen|molecular oxygen]] (O<sub>2</sub>) instead of carbon dioxide ({{CO2}}).<ref name = "Goodsell_2000">{{cite journal | date = November 2000 | vauthors = Goodsell D | title = Rubisco | url = https://pdb101.rcsb.org/motm/11 | journal = Molecule of the Month | publisher = RCSB PDB (Research Collaboratory for Structural Bioinformatics PDB) | doi = 10.2210/rcsb_pdb/mom_2000_11 | url-access = subscription }}</ref>
Discriminating between the substrates {{CO2}} and O<sub>2</sub> is attributed to the differing interactions of the substrate's [[quadrupole moment]]s and a high [[electrostatic field]] [[gradient]].<ref name="Stec_2012" /> This gradient is established by the [[Dimer (chemistry)|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<sub>2</sub> and {{CO2}}. These conditions help explain the low turnover rate found in RuBisCO: In order to increase the strength of the [[electric field]] necessary for sufficient interaction with the substrates' [[quadrupole moment]]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]].<ref>{{cite journal | vauthors = Satagopan S, Spreitzer RJ | title = Plant-like substitutions in the large-subunit carboxy terminus of Chlamydomonas Rubisco increase CO2/O2 specificity | journal = BMC Plant Biology | volume = 8 | page = 85 | date = July 2008 | issue = 1 | pmid = 18664299 | pmc = 2527014 | doi = 10.1186/1471-2229-8-85 | bibcode = 2008BMCPB...8...85S | doi-access = free }}</ref> This isolation has a significant [[Entropy|entropic]] cost, and results in the poor turnover rate.

==== Binding RuBP ====
Carbamylation of the ε-amino group of Lys210 is stabilized by coordination with the {{chem2|Mg(2+)}}.<ref>{{cite journal | vauthors = Lorimer GH, Miziorko HM | title = Carbamate formation on the epsilon-amino group of a lysyl residue as the basis for the activation of ribulosebisphosphate carboxylase by CO2 and Mg2+ | journal = Biochemistry | volume = 19 | issue = 23 | pages = 5321–5328 | date = November 1980 | pmid = 6778504 | doi = 10.1021/bi00564a027 }}</ref> This reaction involves binding of the carboxylate termini of Asp203 and Glu204 to the {{chem2|Mg(2+)}} ion. The substrate RuBP binds {{chem2|Mg(2+)}} displacing two of the three aquo ligands.<ref name="Andersson_2008"/><ref name="genbase">{{cite journal | vauthors = Cleland WW, Andrews TJ, Gutteridge S, Hartman FC, Lorimer GH | title = Mechanism of Rubisco: The Carbamate as General Base | journal = Chemical Reviews | volume = 98 | issue = 2 | pages = 549–562 | date = April 1998 | pmid = 11848907 | doi = 10.1021/cr970010r }}</ref><ref name ="Andersson _1989">{{cite journal | vauthors = Andersson I, Knight S, Schneider G, Lindqvist Y, Lundqvist T, Brändén CI, Lorimer GH |title=Crystal structure of the active site of ribulose-bisphosphate carboxylase |journal=Nature |date=1989 |volume=337 |issue=6204 |pages=229–234|doi=10.1038/337229a0 |bibcode=1989Natur.337..229A |s2cid=4370073 }}</ref>

==== Enolisation ====
[[Keto–enol tautomerism|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,<ref name="genbase"/><ref name="Hartman_1994">{{cite journal | vauthors = Hartman FC, Harpel MR | title = Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphate carboxylase/oxygenase | journal = Annual Review of Biochemistry | volume = 63 | pages = 197–234 | year = 1994 | pmid = 7979237 | doi = 10.1146/annurev.bi.63.070194.001213 }}</ref> but the steric constraints observed in crystal structures have made Lys210 the most likely candidate.<ref name="Andersson_2008"/> 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.<ref name="genbase"/><ref name ="Andersson _1989"/>

==== Carboxylation====
[[File:Crystal structure of active site of RuBisCO bound to 2-Carboxyarabinitol-1,5-Bisphosphate.png|thumb|A 3D image of the active site of spinach RuBisCO complexed with the inhibitor 2-carboxyarabinitol-1,5-bisphosphate, {{CO2}}, and {{chem2|Mg(2+)}}. (PDB: 1IR1; Ligand View [CAP]501:A)]]
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 {{CO2}} substrate as it replaces the third {{chem2|Mg(2+)}}-coordinated water molecule and add directly to the enediol. No Michaelis complex is formed in this process.<ref name="Andersson_2008"/><ref name="Hartman_1994"/> Hydration of this ketone results in an additional hydroxy group on C3, forming a [[Geminal diol|gem-diol]] intermediate.<ref name="genbase"/><ref name="and1997">{{cite journal | vauthors = Taylor TC, Andersson I | title = The structure of the complex between rubisco and its natural substrate ribulose 1,5-bisphosphate | journal = Journal of Molecular Biology | volume = 265 | issue = 4 | pages = 432–444 | date = January 1997 | pmid = 9034362 | doi = 10.1006/jmbi.1996.0738 }}</ref> Carboxylation and hydration have been proposed as either a single concerted step<ref name="genbase"/> or as two sequential steps.<ref name="and1997"/> 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.<ref name="Andersson_2008"/>

==== 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 charged carboxylate.<ref name="Andersson_2008"/> 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.<ref name="Andersson_2008"/>

===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]]. This product, also known as 3-phosphoglycerate, can be used to produce larger molecules such as [[glucose]].

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]], which involves enzymes and cytochromes located in the [[mitochondria]] and [[peroxisomes]] (this is a case of [[Metabolite damage and its repair or pre-emption|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]]. 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 [[C4 carbon fixation|{{C4}} carbon fixation]], [[crassulacean acid metabolism]], and the use of [[pyrenoid]].

Rubisco [[Enzyme promiscuity|side activities]] can lead to useless or inhibitory by-products. Important inhibitory by-products include [[xylulose 1,5-bisphosphate]] 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 {{CO2}} and RuBP concentrations and solved by Rubisco activase (see below).<ref>{{cite journal | vauthors = Pearce FG | title = Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies | journal = The Biochemical Journal | volume = 399 | issue = 3 | pages = 525–534 | date = November 2006 | pmid = 16822231 | pmc = 1615894 | doi = 10.1042/BJ20060430 }}</ref>

===Rate of enzymatic activity===
[[Image:Calvin-cycle4.svg|thumb|upright=1.35|Overview of the [[Calvin cycle]] and carbon fixation.]]

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.<ref>{{cite journal | vauthors = Ellis RJ | title = Biochemistry: Tackling unintelligent design | journal = Nature | volume = 463 | issue = 7278 | pages = 164–165 | date = January 2010 | pmid = 20075906 | doi = 10.1038/463164a | s2cid = 205052478 | bibcode = 2010Natur.463..164E }}</ref> 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 (fluid)|stroma]] rises from 7.0 to 8.0 because of the proton (hydrogen ion, {{chem2|H+}}) gradient created across the [[thylakoid]] membrane. The movement of protons into thylakoids is [[Light-dependent reaction|driven by light]] and is fundamental to [[ATP synthase#Plant ATP synthase|ATP synthesis]] in chloroplasts ''(Further reading: [[Photosynthetic reaction centre]]; [[Light-dependent reactions]])''. To balance ion potential across the membrane, magnesium ions ({{chem2|Mg(2+)}}) 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, {{GO|GO:0046863}}, {{UniProt|P10896}}), is required to allow the rapid formation of the critical [[carbamate]] in the active site of RuBisCO.<ref name="pmid16245090">{{cite journal | vauthors = Portis AR | title = Rubisco activase - Rubisco's catalytic chaperone | journal = Photosynthesis Research | volume = 75 | issue = 1 | pages = 11–27 | year = 2003 | pmid = 16245090 | doi = 10.1023/A:1022458108678 | bibcode = 2003PhoRe..75...11P | s2cid = 2632 }}</ref><ref name="pmid15236471">{{cite journal | vauthors = Jin SH, Jiang DA, Li XQ, Sun JW | title = Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene | journal = Journal of Zhejiang University Science | volume = 5 | issue = 8 | pages = 897–899 | date = August 2004 | pmid = 15236471 | doi = 10.1631/jzus.2004.0897 | doi-broken-date = 26 October 2025 | s2cid = 1496584 }}</ref> This is required because [[ribulose 1,5-bisphosphate]] (RuBP) binds more strongly to the active sites of RuBisCO when excess carbamate is present, preventing processes from 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]] (CA1P).<ref name="pmid7818481">{{cite journal | vauthors = Andralojc PJ, Dawson GW, Parry MA, Keys AJ | title = Incorporation of carbon from photosynthetic products into 2-carboxyarabinitol-1-phosphate and 2-carboxyarabinitol | journal = The Biochemical Journal | volume = 304 | issue = 3 | pages = 781–786 | date = December 1994 | pmid = 7818481 | pmc = 1137402 | doi = 10.1042/bj3040781 }}</ref> 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 [[Chemical conformation|conformation]] that is protected from [[protease|proteolysis]].<ref>{{cite journal | vauthors = Khan S, Andralojc PJ, Lea PJ, Parry MA | title = 2'-carboxy-D-arabitinol 1-phosphate protects ribulose 1, 5-bisphosphate carboxylase/oxygenase against proteolytic breakdown | journal = European Journal of Biochemistry | volume = 266 | issue = 3 | pages = 840–847 | date = December 1999 | pmid = 10583377 | doi = 10.1046/j.1432-1327.1999.00913.x | doi-access = free }}</ref> 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]]. 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.<ref name="pmid11706186">{{cite journal | vauthors = Salvucci ME, Osteryoung KW, Crafts-Brandner SJ, Vierling E | title = Exceptional sensitivity of Rubisco activase to thermal denaturation in vitro and in vivo | journal = Plant Physiology | volume = 127 | issue = 3 | pages = 1053–1064 | date = November 2001 | pmid = 11706186 | pmc = 129275 | doi = 10.1104/pp.010357 }}</ref><ref name="pmid11069297">{{cite journal | vauthors = Crafts-Brandner SJ, Salvucci ME | title = Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 24 | pages = 13430–13435 | date = November 2000 | pmid = 11069297 | pmc = 27241 | doi = 10.1073/pnas.230451497 | doi-access = free | bibcode = 2000PNAS...9713430C }}</ref>


====By activase====
The removal of the inhibitory RuBP, CA1P, and the other inhibitory substrate analogs by activase requires the consumption of [[Adenosine triphosphate|ATP]]. This reaction is inhibited by the presence of [[Adenosine diphosphate|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]]) state through another small regulatory protein, [[thioredoxin]]. 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.<ref>{{cite journal | vauthors = Zhang N, Kallis RP, Ewy RG, Portis AR | title = Light modulation of Rubisco in Arabidopsis requires a capacity for redox regulation of the larger Rubisco activase isoform | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 99 | issue = 5 | pages = 3330–3334 | date = March 2002 | pmid = 11854454 | pmc = 122518 | doi = 10.1073/pnas.042529999 | doi-access = free | bibcode = 2002PNAS...99.3330Z }}</ref>

====By phosphate====
In cyanobacteria, inorganic [[phosphate]] (P<sub>i</sub>) also participates in the co-ordinated regulation of photosynthesis: P<sub>i</sub> 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<sub>i</sub> levels, which might cause it to act in a similar way to how RuBisCO activase functions in higher plants.<ref>{{cite journal | vauthors = Marcus Y, Gurevitz M | title = Activation of cyanobacterial RuBP-carboxylase/oxygenase is facilitated by inorganic phosphate via two independent mechanisms | journal = European Journal of Biochemistry | volume = 267 | issue = 19 | pages = 5995–6003 | date = October 2000 | pmid = 10998060 | doi = 10.1046/j.1432-1327.2000.01674.x | doi-access = free }}</ref>

====By carbon dioxide====
Since carbon dioxide and oxygen [[Enzyme inhibitor|compete]] 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]]). Several times during the evolution of plants, mechanisms have evolved for increasing the level of carbon dioxide in the stroma (see [[C4 carbon fixation|{{C4}} 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]], such that healthy leaves in bright light may have zero net carbon fixation when the ratio of O<sub>2</sub> to {{CO2}} available to RuBisCO shifts too far towards oxygen. This phenomenon is primarily temperature-dependent: high temperatures can decrease the concentration of {{CO2}} dissolved in the moisture of leaf tissues. This phenomenon is also related to [[Water scarcity|water stress]]: since plant leaves are evaporatively cooled, limited water causes high leaf temperatures. [[C4 plants|{{C4}} plants]] use the enzyme [[C4 carbon fixation|PEP carboxylase]] initially, which has a higher affinity for {{CO2}}. The process first makes a 4-carbon intermediate compound, hence the name {{C4}} plants, which is shuttled into a site of [[C3 plants|{{C3}} photosynthesis]] then decarboxylated, releasing {{CO2}} to boost the concentration of {{CO2}}.

[[Crassulacean acid metabolism]] (CAM) plants keep their [[stoma]]ta closed during the day, which conserves water but prevents the light-independent reactions (a.k.a. the [[Calvin Cycle]]) from taking place, since these reactions require {{CO2}} to pass by gas exchange through these openings. Evaporation through the upper side of a leaf is prevented by a layer of [[wax]].

== Genetic engineering ==

Since RuBisCO is often rate-limiting for photosynthesis in plants, it may be possible to improve [[photosynthetic efficiency]] by modifying RuBisCO genes in plants to increase catalytic activity and/or decrease oxygenation rates.<ref>{{cite journal | vauthors = Spreitzer RJ, Salvucci ME | title = Rubisco: structure, regulatory interactions, and possibilities for a better enzyme | journal = Annual Review of Plant Biology | volume = 53 | pages = 449–475 | year = 2002 | issue = 1 | pmid = 12221984 | doi = 10.1146/annurev.arplant.53.100301.135233 | bibcode = 2002AnRPB..53..449S | s2cid = 9387705 }}</ref><ref name="John_Timmer_2017">{{cite web|url=https://arstechnica.com/science/2017/12/key-plant-proteins-that-grab-co%e2%82%82-finally-made-in-bacteria/| vauthors = Timmer J |title=We may now be able to engineer the most important lousy enzyme on the planet|date=7 December 2017|work=Ars Technica|access-date=5 January 2019|name-list-style=vanc}}</ref><ref name="John_Timmer_2019">{{cite web|url=https://arstechnica.com/science/2019/01/re-engineering-photosynthesis-gives-plants-a-40-growth-boost/| vauthors = Timmer J |title=Fixing photosynthesis by engineering it to recycle a toxic mistake|date=3 January 2019|work=Ars Technica|access-date=5 January 2019|name-list-style=vanc}}</ref><ref name="South_2019">{{cite journal | vauthors = South PF, Cavanagh AP, Liu HW, Ort DR | title = Synthetic glycolate metabolism pathways stimulate crop growth and productivity in the field | journal = Science | volume = 363 | issue = 6422 | article-number = eaat9077 | date = January 2019 | pmid = 30606819 | pmc = 7745124 | doi = 10.1126/science.aat9077 | doi-access = free }}</ref> This could improve [[Carbon sequestration|sequestration of {{CO2}}]] and be a strategy to increase crop yields.<ref name="Furbank_2015">{{Cite journal | vauthors = Furbank RT, Quick WP, Sirault XR |title=Improving photosynthesis and yield potential in cereal crops by targeted genetic manipulation: Prospects, progress and challenges|journal=Field Crops Research|volume=182|pages=19–29|doi=10.1016/j.fcr.2015.04.009|year=2015|bibcode=2015FCrRe.182...19F |doi-access=free}}</ref> 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 [[plastome|chloroplast DNA]], and altering RuBisCO genes to increase specificity for carbon dioxide or otherwise increase the rate of carbon fixation.<ref name = "Parry_2003">{{cite journal | vauthors = Parry MA, Andralojc PJ, Mitchell RA, Madgwick PJ, Keys AJ | title = Manipulation of Rubisco: the amount, activity, function and regulation | journal = Journal of Experimental Botany | volume = 54 | issue = 386 | pages = 1321–1333 | date = May 2003 | pmid = 12709478 | doi = 10.1093/jxb/erg141 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Ogbaga CC, Stepien P, Athar HU, Ashraf M | title = Engineering Rubisco activase from thermophilic cyanobacteria into high-temperature sensitive plants | journal = Critical Reviews in Biotechnology | volume = 38 | issue = 4 | pages = 559–572 | date = June 2018 | pmid = 28937283 | doi = 10.1080/07388551.2017.1378998 | s2cid = 4191791 }}</ref>

=== Mutagenesis in plants ===
In general, [[site-directed mutagenesis]] of RuBisCO has been mostly unsuccessful,<ref name="Furbank_2015" /> though mutated forms of the protein have been achieved in tobacco plants with subunit C<sub>4</sub> species,<ref>{{cite journal | vauthors = Whitney SM, Sharwood RE, Orr D, White SJ, Alonso H, Galmés J | title = Isoleucine 309 acts as a C4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 35 | pages = 14688–14693 | date = August 2011 | pmid = 21849620 | pmc = 3167554 | doi = 10.1073/pnas.1109503108 | doi-access = free | bibcode = 2011PNAS..10814688W }}</ref> and a RuBisCO with more C<sub>4</sub>-like kinetic characteristics have been attained in rice via nuclear transformation.<ref>{{cite journal | vauthors = Ishikawa C, Hatanaka T, Misoo S, Miyake C, Fukayama H | title = Functional incorporation of sorghum small subunit increases the catalytic turnover rate of Rubisco in transgenic rice | journal = Plant Physiology | volume = 156 | issue = 3 | pages = 1603–1611 | date = July 2011 | pmid = 21562335 | pmc = 3135941 | doi = 10.1104/pp.111.177030 }}</ref> Robust and reliable engineering for yield of RuBisCO and other enzymes in the C<sub>3</sub> cycle was shown to be possible,<ref>{{Cite book| vauthors = Stracquadanio G, Umeton R, Papini A, Lio P, Nicosia G |title=2010 IEEE International Conference on BioInformatics and BioEngineering |chapter=Analysis and Optimization of C3 Photosynthetic Carbon Metabolism |date=2010|location=Philadelphia, PA, USA|publisher=IEEE|pages=44–51|doi=10.1109/BIBE.2010.17|hdl=1721.1/101094 |isbn=978-1-4244-7494-3|s2cid=5568464|hdl-access=free}}</ref> and it was first achieved in 2019 through a synthetic biology approach.<ref name="South_2019" />

One avenue is to introduce RuBisCO variants with naturally high specificity values such as the ones from the [[red alga]] ''Galdieria partita'' into plants. This may improve the photosynthetic efficiency of crop plants, although possible negative impacts have yet to be studied.<ref>{{cite journal | vauthors = Whitney SM, Andrews TJ | title = Plastome-encoded bacterial ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 98 | issue = 25 | pages = 14738–14743 | date = December 2001 | pmid = 11724961 | pmc = 64751 | doi = 10.1073/pnas.261417298 | doi-access = free | bibcode = 2001PNAS...9814738W }}</ref> Advances in this area include the replacement of the tobacco enzyme with that of the purple photosynthetic bacterium ''[[Rhodospirillum rubrum]]''.<ref>{{cite journal | vauthors = John Andrews T, Whitney SM | title = Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants | journal = Archives of Biochemistry and Biophysics | volume = 414 | issue = 2 | pages = 159–169 | date = June 2003 | pmid = 12781767 | doi = 10.1016/S0003-9861(03)00100-0 }}</ref> In 2014, two transplastomic tobacco lines with functional RuBisCO from the [[cyanobacterium]] ''[[Synechococcus 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 {{CO2}} fixation rates when measured as carbon molecules per RuBisCO. However, the mutant plants grew more slowly than wild-type.<ref>{{cite journal | vauthors = Lin MT, Occhialini A, Andralojc PJ, Parry MA, Hanson MR | title = A faster Rubisco with potential to increase photosynthesis in crops | journal = Nature | volume = 513 | issue = 7519 | pages = 547–550 | date = September 2014 | pmid = 25231869 | pmc = 4176977 | doi = 10.1038/nature13776 | bibcode = 2014Natur.513..547L }}</ref>

A recent theory explores the trade-off between the relative specificity (i.e., ability to favour {{CO2}} fixation over O<sub>2</sub> incorporation, which leads to the energy-wasteful process of [[photorespiration]]) 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.<ref name=Tcherkez06>{{cite journal | vauthors = Tcherkez GG, Farquhar GD, Andrews TJ | title = Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 103 | issue = 19 | pages = 7246–7251 | date = May 2006 | pmid = 16641091 | pmc = 1464328 | doi = 10.1073/pnas.0600605103 | doi-access = free | bibcode = 2006PNAS..103.7246T }}</ref><ref>{{Cite journal |last1=Savir |first1=Yonatan |last2=Noor |first2=Elad |last3=Milo |first3=Ron |last4=Tlusty |first4=Tsvi |date=2010-02-23 |title=Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape |journal=Proceedings of the National Academy of Sciences |volume=107 |issue=8 |pages=3475–3480 |doi=10.1073/pnas.0911663107 |doi-access=free |pmc=2840432 |pmid=20142476 |arxiv=1007.4461 |bibcode=2010PNAS..107.3475S }}</ref> It has been also suggested that the oxygenase reaction of RuBisCO prevents {{CO2}} depletion near its active sites and provides the maintenance of the chloroplast redox state.<ref>{{cite journal | vauthors = Igamberdiev AU | title = Control of Rubisco function via homeostatic equilibration of CO2 supply | journal = Frontiers in Plant Science | volume = 6 | page = 106 | date = 2015 | pmid = 25767475 | pmc = 4341507 | doi = 10.3389/fpls.2015.00106 | doi-access = free }}</ref>

Since photosynthesis is the single most effective natural regulator of [[carbon dioxide in the Earth's atmosphere]],<ref>{{cite journal | vauthors = Igamberdiev AU, Lea PJ | title = Land plants equilibrate O2 and CO2 concentrations in the atmosphere | journal = Photosynthesis Research | volume = 87 | issue = 2 | pages = 177–194 | date = February 2006 | pmid = 16432665 | doi = 10.1007/s11120-005-8388-2 | bibcode = 2006PhoRe..87..177I | s2cid = 10709679 }}</ref> 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]]s as unfolded proteins.<ref>{{cite journal | vauthors = Bracher A, Whitney SM, Hartl FU, Hayer-Hartl M | title = Biogenesis and Metabolic Maintenance of Rubisco | journal = Annual Review of Plant Biology | volume = 68 | pages = 29–60 | date = April 2017 | issue = 1 | pmid = 28125284 | doi = 10.1146/annurev-arplant-043015-111633 | bibcode = 2017AnRPB..68...29B | doi-access = free }}</ref><ref>{{cite journal | vauthors = Sjuts I, Soll J, Bölter B | title = Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms | language = English | journal = Frontiers in Plant Science | volume = 8 | page = 168 | date = 2017 | pmid = 28228773 | pmc = 5296341 | doi = 10.3389/fpls.2017.00168 | bibcode = 2017FrPS....8..168S | doi-access = free }}</ref> Furthermore, sufficient expression and interaction with Rubisco activase are major challenges as well.<ref name = "Parry_2003" /> One successful method for expression of Rubisco in [[Escherichia coli|''E. coli'']] involves the co-expression of multiple chloroplast chaperones, though this has only been shown for ''[[Arabidopsis thaliana]]'' Rubisco.<ref>{{cite journal | vauthors = Aigner H, Wilson RH, Bracher A, Calisse L, Bhat JY, Hartl FU, Hayer-Hartl M | title = Plant RuBisCo assembly in ''E. coli'' with five chloroplast chaperones including BSD2 | journal = Science | volume = 358 | issue = 6368 | pages = 1272–1278 | date = December 2017 | pmid = 29217567 | doi = 10.1126/science.aap9221 | doi-access = free | bibcode = 2017Sci...358.1272A | hdl = 11858/00-001M-0000-002E-8B4D-B | hdl-access = free }}</ref>

== 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]]s, [[kinase]]s, and regulatory proteins found in lower abundance (10-100 molecules per cell) within plants.<ref name="Heazlewood_2012">{{Cite book|title=Proteomic applications in biology| vauthors = Heazlewood J |publisher=InTech Manhattan|year=2012|isbn=978-953-307-613-3|location=New York}}</ref> For example, using [[mass spectrometry]] 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]] solution.<ref>{{cite book | vauthors = Gupta R, Kim ST | title = Proteomic Profiling | chapter = Depletion of RuBisCO Protein Using the Protamine Sulfate Precipitation Method | series = Methods in Molecular Biology | volume = 1295 | pages = 225–33 | date = 2015 | pmid = 25820725 | doi = 10.1007/978-1-4939-2550-6_17 | publisher = Humana Press | location = New York, NY | isbn = 978-1-4939-2549-0 }}</ref> Other existing methods for depleting RuBisCO and studying lower abundance proteins include [[fractionation]] techniques with calcium and phytate,<ref>{{cite journal | vauthors = Krishnan HB, Natarajan SS | title = A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins | journal = Phytochemistry | volume = 70 | issue = 17–18 | pages = 1958–1964 | date = December 2009 | pmid = 19766275 | doi = 10.1016/j.phytochem.2009.08.020 | bibcode = 2009PChem..70.1958K }}</ref> [[gel electrophoresis]] with polyethylene glycol,<ref>{{cite journal | vauthors = Kim ST, Cho KS, Jang YS, Kang KY | title = Two-dimensional electrophoretic analysis of rice proteins by polyethylene glycol fractionation for protein arrays | journal = Electrophoresis | volume = 22 | issue = 10 | pages = 2103–2109 | date = June 2001 | pmid = 11465512 | doi = 10.1002/1522-2683(200106)22:10<2103::aid-elps2103>3.0.co;2-w | s2cid = 38878805 }}</ref><ref>{{cite journal | vauthors = Xi J, Wang X, Li S, Zhou X, Yue L, Fan J, Hao D | title = Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome | journal = Phytochemistry | volume = 67 | issue = 21 | pages = 2341–2348 | date = November 2006 | pmid = 16973185 | doi = 10.1016/j.phytochem.2006.08.005 | bibcode = 2006PChem..67.2341X }}</ref> [[affinity chromatography]],<ref>{{cite journal | vauthors = Cellar NA, Kuppannan K, Langhorst ML, Ni W, Xu P, Young SA | title = Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase | journal = Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences | volume = 861 | issue = 1 | pages = 29–39 | date = January 2008 | pmid = 18063427 | doi = 10.1016/j.jchromb.2007.11.024 }}</ref><ref>{{cite journal | vauthors = Agrawal GK, Jwa NS, Rakwal R | title = Rice proteomics: ending phase I and the beginning of phase II | journal = Proteomics | volume = 9 | issue = 4 | pages = 935–963 | date = February 2009 | pmid = 19212951 | doi = 10.1002/pmic.200800594 | s2cid = 2455432 }}</ref> and aggregation using [[Dithiothreitol|DTT]],<ref>{{Cite journal | vauthors = Cho JH, Hwang H, Cho MH, Kwon YK, Jeon JS, Bhoo SH, Hahn TR |date= July 2008 |title= The effect of DTT in protein preparations for proteomic analysis: Removal of a highly abundant plant enzyme, ribulose bisphosphate carboxylase/oxygenase|journal=Journal of Plant Biology|language=en|volume=51|issue=4|pages=297–301|doi=10.1007/BF03036130|bibcode= 2008JPBio..51..297C |s2cid= 23636617 |issn=1226-9239}}</ref> though these methods are more time-consuming and less efficient when compared to protamine sulfate precipitation.<ref name="Heazlewood_2012" />

== 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 (genetics)|locus]] for analysis of [[phylogenetics]] in [[plant taxonomy]].<ref>{{cite journal | vauthors = Chase MW, Soltis DE, Olmstead RG, Morgan D, Les DH, Mishler BD, etal |title=Phylogenetics of Seed Plants: An Analysis of Nucleotide Sequences from the Plastid Gene ''rbc''L|journal=[[Annals of the Missouri Botanical Garden]]|date=1993|volume=80|issue=3|pages=528–580|doi=10.2307/2399846|ref={{harvid|Chase et al|1993}}|jstor=2399846|bibcode=1993AnMBG..80..528C |url=https://spectrum.library.concordia.ca/6741/1/Dayanandan_AnnalsMissouriBotanicalGardens_1993.pdf|hdl=1969.1/179875|hdl-access=free}}</ref>

=== Origin ===
{{missing information|section|explanation of the large-only oligomeric forms up to (L<sub>2</sub>)<sub>5</sub>; explanation of what the small subunit probably does (improve CO2/O2 discrimination); maybe a {{tlx|external image}} pointing to the MotM and Erb 2018 pics|date=March 2022}}
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]]''. This bacterium has a rbcL-like protein with a [[2,3-diketo-5-methylthiopentyl-1-phosphate enolase]] function, part of the [[methionine salvage pathway]].<ref>{{cite journal | vauthors = Ashida H, Saito Y, Nakano T, Tandeau de Marsac N, Sekowska A, Danchin A, Yokota A | title = RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO | journal = Journal of Experimental Botany | volume = 59 | issue = 7 | pages = 1543–1554 | date = 19 June 2007 | pmid = 18403380 | doi = 10.1093/jxb/ern104 | doi-access = free }}</ref> 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.<ref name = "Erb_2018" /> The small subunit probably first evolved in anaerobic and thermophilic organisms, where it enabled RuBisCO to catalyze its reaction at higher temperatures.<ref>{{cite journal |last1=Schulz |first1=L |last2=Guo |first2=Z |last3=Zarzycki |first3=J |last4=Steinchen |first4=W |last5=Schuller |first5=JM |last6=Heimerl |first6=T |last7=Prinz |first7=S |last8=Mueller-Cajar |first8=O |last9=Erb |first9=TJ |last10=Hochberg |first10=GKA |title=Evolution of increased complexity and specificity at the dawn of form I Rubiscos |journal=Science |date=2022-10-14 |volume=378 |issue=6616 |pages=155–160 |doi=10.1126/science.abq1416 |pmid=36227987|bibcode=2022Sci...378..155S |s2cid=252897276 }}</ref> In addition to its effect on stabilizing catalysis, it enabled the evolution of higher specificities for {{CO2}} over O<sub>2</sub> 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.

=== C<sub>4</sub> ===
With the mass convergent evolution of the [[C4 carbon fixation|C<sub>4</sub>-fixation pathway]] in a diversity of plant lineages, ancestral C<sub>3</sub>-type RuBisCO evolved to have faster turnover of {{CO2}} in exchange for lower specificity as a result of the greater localization of {{CO2}} from the [[mesophyll cell]]s into the [[bundle sheath cells]].<ref>{{cite journal | vauthors = Sage RF, Sage TL, Kocacinar F | title = Photorespiration and the evolution of C4 photosynthesis | journal = Annual Review of Plant Biology | volume = 63 | pages = 19–47 | date = 2012 | issue = 1 | pmid = 22404472 | doi = 10.1146/annurev-arplant-042811-105511 | bibcode = 2012AnRPB..63...19S | s2cid = 24199852 }}</ref> This was achieved through enhancement of conformational flexibility of the "open-closed" transition in the [[Calvin Cycle|Calvin cycle]]. 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]]s for C<sub>4</sub> RuBisCO.<ref name="Studer_2014">{{cite journal | vauthors = Studer RA, Christin PA, Williams MA, Orengo CA | title = Stability-activity tradeoffs constrain the adaptive evolution of RubisCO | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 6 | pages = 2223–2228 | date = February 2014 | pmid = 24469821 | pmc = 3926066 | doi = 10.1073/pnas.1310811111 | doi-access = free | bibcode = 2014PNAS..111.2223S }}</ref> Moreover, in order to sustain the destabilizing mutations, the evolution to C<sub>4</sub> 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<sub>4</sub> 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<sub>4</sub> mutations on RuBisCO has been sustained by environmental pressures such as low {{CO2}} concentrations, requiring a sacrifice of stability for new adaptive functions.<ref name="Studer_2014" />

==History of the term==
The term "RuBisCO" was coined humorously in 1979, by [[David Eisenberg]] at a seminar honouring the retirement of the early, prominent RuBisCO researcher, [[Sam Wildman]], and also alluded to the snack food trade name "[[Nabisco]]" in reference to Wildman's attempts to create an edible protein supplement from tobacco leaves.<ref>{{cite journal | vauthors = Wildman SG | title = Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase) | journal = Photosynthesis Research | volume = 73 | issue = 1–3 | pages = 243–250 | year = 2002 | pmid = 16245127 | doi = 10.1023/A:1020467601966 | bibcode = 2002PhoRe..73..243W | s2cid = 7622999 }}</ref><ref>{{cite journal | vauthors = Portis AR, Parry MA | title = Discoveries in Rubisco (Ribulose 1,5-bisphosphate carboxylase/oxygenase): a historical perspective | journal = Photosynthesis Research | volume = 94 | issue = 1 | pages = 121–143 | date = October 2007 | pmid = 17665149 | doi = 10.1007/s11120-007-9225-6 | bibcode = 2007PhoRe..94..121P | s2cid = 39767233 }}</ref>

The capitalization of the name has been long debated. It can be capitalized for each letter of the full name ('''R'''ib'''u'''lose-1,5 '''bis'''phosphate '''c'''arboxylase/'''o'''xygenase), but it has also been argued that is should all be in lower case (rubisco), similar to other terms like scuba or laser.<ref name="Sharkey_2018" />

== See also ==
{{Col-begin}}
{{Col-1-of-2}}
*[[Carbon cycle]]
*[[Photorespiration]]
*[[Pyrenoid]]
*[[C3 carbon fixation]]
{{Col-2-of-2}}
*[[C4 carbon fixation]]
*[[Crassulacean acid metabolism]]/CAM photosynthesis
*[[Carboxysome]]
{{col-end}}

== References ==
{{notelist}}
{{Reflist|32em}}

[[Image:RuBisCOL2S2.png|thumb|right|''Figure 3''. In this figure, each protein chain in the (LS)<sub>2</sub> complex is given its own color for easy identification.]]

== Further reading ==
{{refbegin}}
* {{cite journal | vauthors = Marcus Y, Altman-Gueta H, Finkler A, Gurevitz M | title = Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis | journal = Journal of Bacteriology | volume = 187 | issue = 12 | pages = 4222–4228 | date = June 2005 | pmid = 15937184 | pmc = 1151729 | doi = 10.1128/JB.187.12.4222-4228.2005 }}
* {{cite journal | vauthors = Sugawara H, Yamamoto H, Shibata N, Inoue T, Okada S, Miyake C, Yokota A, Kai Y | display-authors = 6 | title = Crystal structure of carboxylase reaction-oriented ribulose 1, 5-bisphosphate carboxylase/oxygenase from a thermophilic red alga, Galdieria partita | journal = The Journal of Biological Chemistry | volume = 274 | issue = 22 | pages = 15655–15661 | date = May 1999 | pmid = 10336462 | doi = 10.1074/jbc.274.22.15655 | doi-access = free }}
{{refend}}

== External links ==
* {{cite web | vauthors = Gerritsen VB | date = September 2003 | url = http://www.expasy.org/spotlight/back_issues/sptlt038.shtml | title = The Plant Kingdom's sloth | work = Protein Spotlight | publisher = Swiss Institute of Bioinformatics (SIB) | quote = Rubisco plods along at a mere three molecules per second... To bypass such slothfulness, plants synthesize a gross amount of Rubisco, sometimes up to 50% of their total protein content! }}

{{Carbon-carbon lyases}}
{{Enzymes}}

[[Category:Photosynthesis]]
[[Category:EC 4.1.1]]