## 8 Mechanism of the Rubisco Carboxylation Reaction: The Role of Broensted Acid-Base Catalysis  
By integrating information from the body of existing literature concerning crystallographic and mutational studies, [PERSON] et al., proposed in 1998 the first consensus mechanism for the Rubisco carboxylase reaction [40]. The model encompasses RuBP-endiolate coordination to the Mg\({}^{2+}\) ion undergoing subsequent transformation implying, in specific steps, proton-transfer reactions and water addition. From this study it became clear that elucidation of the Rubisco carboxylation mechanism requires large reliance on computational methods, as elementary steps consisting in proton exchanges and water addition cannot be disclosed by analysis of X-ray crystal structures. A decade later, [PERSON] et al. [37] were able to propose a detailed reaction mechanism for the Rubisco carboxylase reaction by integrating computational studies with a density functional theory calculation on active-site fragment models. To allow DFT analysis, active site fragment models of a size of up to 77 atoms were built by using the coordinates of Rubisco crystal structures. In particular the authors have used the crystal structure of the activated spinach Rubisco complexed with Ca\({}^{2+}\) and RuBP (PDB code number 1 RXO [33] showing Ca\({}^{2+}\) coordination as depicted in Figure 9) and that of spinach Rubisco complexed with Mg\({}^{2+}\) and 2 CABP (PDB code number 8 RUC [35]). The building of approximately twenty fragment models enabled mapping of structures of reaction intermediates, and the computational analysis (based on the use of the B3 LYP functional) allowed optimizing of their geometries. Validation of the optimized model structures was carried out comparing calculated interatomic distances with the active-site structures revealed by X-ray diffraction. For 75% of the compared distances, deviation of less than 0.6 A was obtained. Relevant features of the reaction mechanism as proposed by [PERSON] et al., in 2008 [37] are shown in Figure 10 (amino acid sequence numbering refers to Rubisco from spinach). The initial model fragment along the reaction coordinate shown in Figure 10 was modelled using the active site structure shown in Figure 9, but replacing Ca\({}^{2+}\) with Mg\({}^{2+}\). However, Figure 10 does not show the pseudo-octahedral coordination around Mg\({}^{2+}\) as, for a simplified view, only the RuBP ligand and the water molecule (H\({}_{\text{w1}}\)-O\({}_{\text{w}}\)-H\({}_{\text{w2}}\)) are highlighted. According to [PERSON]'s description, every single step of the reaction involves proton-transfer process. Therefore, amino acid residues acting as Lewis base are highlighted in blue boxes, while amino acid residues stabilizing anions (or the negative charge of dipolar groups) through hydrogen bond are shown in red boxes. Figure 9: Structure of the active site of activated RuBP-bound Rubisco plotted from the 1 RXO PDB file (Protein Data Bank) showing the coordination environment of Ca\({}^{2+}\). (Figure 9 from ref. [33], adapted and used by permission of Elsevier (copyright © 1997)). Relevant steps involving acid-base catalysis are summarized below:  
1. [label=(1)]
2. Enolization step: Following RuBP binding to Mg\({}^{2+}\) through O2 and O3, Lys175-H\({}^{+}\) is proposed to engage in hydrogen bonding with the carbonyl C2=O2. The carbamoylI moiety Lys201-COO\({}^{-}\) (also noted as KCX201) acts as a base abstracting the C3-H3 proton allowing RuBP enolization to the 2,3-_cis_-enediol(ate) form (shown in structure I). Both KCX201-H and LYS175-H\({}^{+}\) are proposed to engage in hydrogen bonding with O2 partially neutralizing its negative charge. His294 is proposed to play an important role in RuBP enolization, engaging in hydrogen bonding with H-O3 and preventing, thus, its interaction with KCX201\({}^{-}\). As a matter of fact, it is assumed that in the absence of the His294-H-O3 interaction the carbamoylI group would abstract H-O3 more easily than H3-C3. 3. [label=(2)]
3.