**6. Phosphorus in key enzymes of the dark phase**

Several key enzymes in the Calvin-Benson cycle contain P as a major structural component, highlighting the importance of P in the dark phase of photosynthesis. These enzymes include RuBisCO, PGK, GAPDH, and others involved in the regeneration stage of the cycle [50].

#### **6.1 RuBisCO**

RuBisCO, the most abundant enzyme on Earth, is responsible for the carboxylation of RuBP [25]. The enzyme contains a large subunit and a small subunit, both of which are encoded by the chloroplast genome [51]. The large subunit contains a conserved lysine residue that forms a Schiff base with the phosphate group of RuBP, facilitating the binding of CO2 [52]. The active site of RuBisCO also contains a tightly bound Mg2+ ion, which coordinates with the phosphate groups of RuBP and stabilizes the transition state during carboxylation [53].

#### **6.2 PGK and GAPDH**

Phosphoribuloseglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are responsible for the phosphorylation and reduction of 3-PGA, respectively [29]. Both enzymes contain P as part of their active sites,

with PGK utilizing a phosphohistidine intermediate in its catalytic mechanism, and GAPDH employing a phosphorylated cysteine residue for the transfer of phosphate groups between substrates [29].

#### **6.3 Regeneration enzymes**

The regeneration stage of the Calvin-Benson cycle involves several enzymes that are responsible for the recycling of the CO2 acceptor molecule, RuBP, which is essential for the continued fixation of CO2 by the cycle. Some of these enzymes, such as SBPase and FBPase, contain P in their active sites, which play a critical role in their catalytic activity [54]. For example, FBPase uses a phosphohistidine intermediate in its reaction mechanism, while SBPase utilizes a phosphoserine intermediate. Phosphorus is also an integral component of RuBP itself, which is regenerated during this stage of the cycle [27].
