## 8 Mechanism of the Rubisco Carboxylation Reaction: The Role of Broensted Acid-Base Catalysis
Interestingly, the computational study revealed that the protonation of the C3-gemdiolate from Lys175-H+ (represented as the orange dotted line in Figure 13b) is not favoured, as products are located 5 kcal/mol higher than the C3-gemdiolate intermediate. In contrast, the proton transfer process occurring through the Grothuss mechanism (represented as the blue and green continue lines in Figure 13b) shows an activation energy of 15.6 kcal/mol (in close agreement with experimental data) and obtains products 14 kcal/mol more stable with respect to C3-gemdiolate intermediate. All of the studies carried by the [PERSON] group summarized above indicate that the last reaction steps concerning C2 -C3 scission (of the C3-gemdiolate) and stereospecific C2 protonation described in Figure 13a proceed through preliminary protonation of the P1-group of the C3-gemdiolate. Subsequently, in a concerted reaction mechanism, a proton at the P1-group induces stereospecific protonation of the C2-carbon via the Groththuss mechanism. Due to the rapidly evolving view of the Rubisco reaction mechanism, in a very recent publication, [PERSON] and [PERSON] [43] have considered the reactional events from a completely new perspective. The rationale behind the reaction mechanism proposed in Figure 10 is that RuBP strongly coordinates Mg\({}^{2+}\) and most parts of the Bronsted acid-base reactions imply \"movement\" of amino acid side chains approaching the substrate. As a consequence, the requirement for conformational chain reorganization implies a considerable amount of strain energy. In a very recent publication, [PERSON] and [PERSON] [43] have refined the model of the Rubisco catalytic site using a complex of approximately 340 atoms, as in previous studies, but introducing new structural details. Considering that the structure of Rubisco from spinach (PDB 8 RUC) gives a snapshot of the enzyme in its \"closed\" conformation, a careful analysis of the water molecules identified by X-ray crystallography around RuBP and Mg\({}^{2+}\) suggests the formation of short water wires. The introduction of the \"water wire\" concept into the model allows proton transfer processes to occur through the Grothuss mechanism (at several steps along the reaction coordinate) as an alternative to the previous amino acids side-chain conformation change mechanism. Notably, the new reaction model does not imply a direct coordination of RuBP to Mg\({}^{2+}\). Interestingly, the new model allows the calculation of accurate potential energy values for intermediates and transition states along the reaction coordinate by using the B3 LYP/6-31G* and corresponding corrected B3 LYP-gCP-D3 functional. Figure 14a displays relevant structural features of the initial molecular complex (**A**) considered in the new model. Although the choice of molecular arrangement within the active site is somehow arbitrary, the authors consider this a logical choice based on the information available. Due to the complexity of the overall RuBP carboxylation reaction, only the enolization and carboxylation/hydration steps are analyzed here. Relevant features of Figure 14a are: (i) Mg\({}^{2+}\) is coordinated to the LYS201-carbamylate moiety (usually referred as KCX201) in the initial molecular complex A; (ii) two water molecules are shown engaging in a network of hydrogen bonding with KCX201 and RuBP in A; (iii) His294 is assumed to be unprotonated in the activated RuBP unbound active site, so that it can interact with O3-H once RuBP enters the active site to give the protonated form His294\({}^{+}\). Proton transfer processes required for RuBP enolization are mediated by two proton wires acting in sequence: H3-H\({}_{2}\)O-KCX201 (see transition state A\({}^{\#}\)) and KCX201-H\({}_{2}\)O-H\({}_{2}\)O-O2 (see intermediate B). Enolization is started by assistance of the H3-H\({}_{2}\)O-KCX201 proton wire acting in H3 abstraction to form H\({}_{3}\)O\({}^{+}\) (in A\({}^{\#}\)) followed by H\({}^{+}\) transfer to KCX201 to give KCX201-H in intermediate B.