Atmospheric CO₂ concentration has increased more than 20% during the past 35 years [9]. The major sink for this CO₂ is the approximately 258 billion tons per year that photosynthetic organisms convert into organic carbon compounds through carbon fixation via the Calvin–Benson pathway [10]. This pathway begins with Rubisco (Ribulose 1,5-bisphosphate carboxylase–oxygenase), the most abundant protein on the planet [11].

Rubisco comes in three forms [12]:

Form I, which is found in cyanobacteria, proteobacteria, chlorophyte algae, heterokont algae, and haptophyte algae, and higher plants, is the most common [13,14]. It is a hexadecamer containing eight identical large subunits (~55,000 M_r), each with a metal-binding site, and eight small subunits (~15,000 M_r). The large subunits are coded by a single plastomic gene, whereas the small subunits are coded by a nuclear multigene family that consists of 2 to 22 members, depending on the species [15]. Complex cellular machinery is required to assemble this form of Rubisco and to maintain its activity [16]. Form I Rubisco, until recently, had resisted all efforts to generate a functional holoenzyme in vitro or upon recombinant expression in E. coli [17].

Form II Rubisco, found in proteobacteria, archaea, and dinoflagellate algae, contains one or more isodimers with subunits that share about 30% identity to the large subunit of Form I Rubisco [8].

Form III Rubisco, found in archaea, has one or five isodimers composed of subunits homologous to the large subunit of Form I Rubisco [8].

Form II and Form III Rubisco show greater similarity in their primary sequence to one another than either do to the large subunit of Form I Rubisco [8].

All three forms of Rubisco catalyze not only the reaction in which the carboxylation of the five-carbon sugar RuBP generates two molecules of the three-carbon organic acid 3-phosphoglycerate (3PGA), but also an alternative reaction in which oxidation of RuBP generates one molecule of 3PGA and one of 2PG (Figure 1) [8]. The carboxylation pathway of photosynthesis expends 3 ATP and 2 NADPH per RuBP regenerated and produces a carbon in hexose [18], whereas the oxygenation pathway of photorespiration reportedly expends 3.5 ATP and 2 NADPH per RuBP regenerated but produces no additional organic carbon [19,20].

Rubisco must be activated before it can carboxylate or oxygenate RuBP. Activation of the three forms of Rubisco involves binding of Mn²⁺ or Mg²⁺ [21,22]. Binding of Mg²⁺ requires carbamylation of Rubisco by the addition of CO₂. One histidine at the active site of Rubisco rotates into an alternate conformation, resulting in a transient binding site where Mg²⁺ is partially neutralized by the conversion of two water molecules to hydroxide ions and coordinated indirectly by three histidine residues through the water molecules. Subsequently, the hydroxide ions cause a lysine residue at the active site to become deprotonated and rotate 120 degrees into the trans conformer, which brings its nitrogen into close proximity to the carbon of CO₂, allowing for the formation of a covalent bond that produces a carbamyl group. This carbamyl group causes the Mg²⁺ ion to transfer to a second binding site, after which the histidine that first rotated returns to its original conformation [23]. It is unclear whether binding Mn²⁺ follows a similar mechanism and whether it requires an activator CO₂ to be bound first [21,22]; hence, understanding the mechanism of Mn²⁺ binding to Rubisco is important to future research on Rubisco kinetics. During in vitro studies, Rubisco is often activated at pH 8.0 in the presence of CO₂ and either Mg²⁺ or Mn²⁺.

Rubisco can also bind to other metals. When bound to Fe²⁺, Ni²⁺, Cu²⁺, Ca²⁺, or Co²⁺, Rubisco may exhibit some carboxylase and oxygenase activity [24]. For example, one study found that Rubisco from R. rubrum, when bound to Co²⁺, was incapable of carboxylation but still capable of oxygenation [24]. Another study found that Rubisco from spinach performed both carboxylation and oxygenation when bound to Ni²⁺ or Co²⁺ [25]. When bound to some other metal ions, including Cd²⁺, Cr²⁺, and Ga²⁺, Rubisco cannot catalyze either carboxylation or oxygenation [24]. Although it is known that the metal ion plays a role in stabilizing the activator carbamate and determining the active site’s structure, its effect upon the reactions catalyzed by Rubisco is still not completely understood. One hypothesis is that Mg²⁺, because of its electron-withdrawing properties, polarizes the C2 carbonyl of RuBP, which favors the removal of the C3 proton and thereby contributes to enolization [21].

NADPH complexes strongly with Rubisco and acts as an effector molecule to maintain the Rubisco catalytic pocket in an open confirmation that more rapidly facilitates CO₂-Mg²⁺ activation when CO₂ and Mg²⁺ are present in suboptimal concentrations [26,27,28,29]. The crystal structure of Rubisco with both Mg²⁺ and NADPH as ligands indicates that NADPH binds to the catalytic site of Rubisco through metal-coordinated water molecules [26]. The activated enzyme catalyzes either carboxylation or oxygenation of the enediol form of the five-carbon sugar ribulose-1,5-bisphosphate (RuBP) [14,21,22,30,31].

Several factors alter the balance between Rubisco carboxylation and oxygenation and, thereby, alter the relative rates of photosynthesis and photorespiration. These include the concentrations of CO₂ and O₂ at the active site of Rubisco, the specificity of the enzyme for each gas, and whether the enzyme is associated with Mg²⁺ or Mn²⁺ [32]. These divalent cations share the same binding site in Rubisco [14,22,33], and in tobacco, Rubisco associates with both metals and rapidly exchanges one metal for the other [32]. Nonetheless, nearly all recent studies on the photosynthetic and photorespiratory pathways have been conducted in the presence of Mg²⁺ and absence of Mn²⁺ [8]. Rubisco binding of Mg²⁺ accelerates carboxylation, whereas binding of Mn²⁺ slows carboxylation [25,34,35,36,37,38]. Chloroplastic Mg²⁺ and Mn²⁺ activities seem to be regulated at the cellular level because in isolated tobacco chloroplasts, activities of the metals were directly proportional to their concentrations in the medium [32]. The thermodynamics of binding Mg²⁺ to Rubisco were similar for enzymes isolated from a Form I and a Form II species [32]. By contrast, the thermodynamics of binding differed greatly between the two Rubisco forms when the enzymes were associated with Mn²⁺ [32].

Mg²⁺ and Mn²⁺ have nearly identical ionic radii but highly disparate electron configurations: Mg²⁺ (1s²2s²2p⁶ or [Ne]) has a very stable outer shell [8], whereas Mn²⁺ has five unpaired d electrons (1s²2s²2p⁶3s²3p⁶3d⁵ or [Ar]3d⁵) that are susceptible to many redox reactions. An aerated solution of activated Mn²⁺-Rubisco exhibits a long-lived chemiluminescence when RuBP is added [39,40]. This chemiluminescence was attributed to a spin-flip within the Mn²⁺ 3d manifold, leading to an excited quartet (S = ³/₂) d⁵ electronic configuration that decays over the course of 1 to 5 min back to the sextet (S = ⁵/₂) ground state electronic configuration [39]. Excited states are intrinsically better oxidants and reductants (larger reduction/oxidation potentials) than their corresponding ground states [41,42,43]; thus, the observed chemiluminescence opens the possibility that the RuBP-O₂-Mn²⁺—Rubisco excited state may be quenched via electron transfer. Consequently, the liberated reducing equivalent could participate in the reduction of NADP⁺ to NADPH (Figure 2, blue pathway). In this way, oxidation of RuBP via O₂ may proceed in a spin-allowed manner, while the Mn²⁺ remains “innocent” in the generation of the oxygenated RuBP precursor. Mn²⁺-centered redox may still proceed, with oxidation of excited Mn²⁺ to Mn³⁺ occurring in a manner independent of, but parallel to, substrate oxidation.

In wheat leaves, the ratio of Mn²⁺ to Mg²⁺ contents increased as the CO₂ levels increased and when the nitrogen source was nitrate rather than ammonium [32]. Nitrate assimilation into amino acids in shoots is heavily dependent on photorespiration, whereas ammonium assimilation is much less so. This indicates that plants shifted to Rubisco Mn²⁺ binding in order to compensate for the slower photorespiration rates and slower amino acid production that would otherwise occur under elevated CO₂ and nitrate nutrition.

The 3-phosphoglycerate produced during photorespiration, like that produced during photosynthesis, is converted to triose phosphate and used to regenerate RuBP. On the other hand, 2-phosphoglycolate is converted to glycolate by phosphoglycolate phosphatase. In the peroxisome and mitochondrion, a series of reactions converts glycolate to glycerate, which is ultimately returned to the chloroplast to regenerate RuBP (Figure 2) [8]. In addition to Rubisco, several other chloroplast enzymes in the photorespiratory pathway, including malic enzyme and phosphoglycolate phosphatase, bind either Mg²⁺ or Mn²⁺ [8]. The plastid isoform of malic enzyme in Arabidopsis and tobacco catalyzes the reverse pyruvate synthesis reaction (pyruvate + CO₂ + NADPH → malate + NADP) [44,45]. Phosphoglycolate phosphatase, which is responsible for the hydrolysis of 2-phosphoglycolate to glycolate, binds to and is activated by either metal [46]. Hypothesized is an alternative photorespiratory pathway that increases photorespiration energy efficiency by generating malate (RuBP + O₂ + CO₂ + H₂O → glycolate + malate + 2Pi) when Mn²⁺ binds to these enzymes (Figure 2) [8].

3. Conclusions