## 8 Mechanism of the Rubisco Carboxylation Reaction: The Role of Broensted Acid-Base Catalysis
Carboxyltion step: following RuBP enolization, His294 is proposed to abstract the H3-O3 proton allowing ketonsation at C3 followed by negative charge localisation at C2 (structure I). The _Si_ face of the incipient C2-carbonian attacks CO\({}_{2}\) forming the 2-carboxy-3-keto-D-arabinitol-1,5-bisphosphate intermediate. Structure III evidences the hydrogen-bonding network, stabilizing the geometry of the carboxylate intermediate within the fragment model. Relevant interactions are: (i) the protonated Lys334-H\({}^{+}\) and the water molecule stabilise the negative charge of the carboxylate moiety; (ii) His294-H\({}^{+}\) maintains hydrogen bonding with O3; (iii) Lys175-H\({}^{+}\) and KCX201-H keep in hydrogen bonding with O2 atom. 4. Hydration step: to start the hydration step (structure III) H\({}_{w1}\) is proposed to be abstracted by Glu204\({}^{-}\) anion allowing nucleophilic attack by OH\({}^{-}\) onto the C3 carbon. His294-H\({}^{+}\) assists the hydration reaction by proton transfer to O3 allowing C3 to reach a sp\({}^{3}\)-hybridization to form a six-carbon gem-diolate intermediate (see structure V). Figure 10: Proposed reaction mechanism for the conversion of RuBP at the Rubisco catalytic site. Amino acid residues acting as Lewis base are highlighted in blue boxes, while amino acid residues stabilizing anions through hydrogen bond are highlighted in red boxes. Numbering (I–IX) refers to geometries of local minima along the reaction pathway obtained using [PERSON]’s theoretical model. (Figure from ref. [37], adapted and used by permission of American Chemical Society (copyright © 2008)). * C2-C3 bond scission and C2 protonation: Structure V shows the six-carbon gemodiate intermediate which is proposed to undergo proton abstraction from H-O3 by His294 with ketonization at the C3 carbon followed by C2-C3 bond scission. As a consequence two molecular fragments are produced:(i) one 3-phospho-D-glyceric acid molecule (lower part of the structure); (ii) a C2-carbonation that delocalizes the negative charge over the carboxylate moiety producing an acid-acid intermediate (it is important to note that the C2=CO\({}_{81}\)O\({}_{82}\)\({}^{-}\) fragment is planar), and this charge is stabilized by Lys334-H\({}^{+}\) and Lys175-H\({}^{+}\) (see structure VII). Finally Lys175-H\({}^{+}\) promotes stereospecific protonation of the C2 carbanion to yield the 3-phospho-D-glycerate. Only at this stage does Lys334-H\({}^{+}\) engage in hydrogen bonding with Lys175. The Rubisco reaction model summarized in Figure 10 has been \"reshaped\" by using recently available computational methods (as hybrid semi-empirical (SE) quantum mechanics, molecular mechanics (QM/MM) plus molecular dynamics (MD) calculations (QN/MM + MD)] in conjunction with energy-minimization calculations. The new computational methods allowed the building of active-site fragment models (FM21 shown in Figure 11) consisting of many hundreds of atoms [35]. FM21 was built based on coordinates from the crystal structure of spinach (PDB code 8 RUC, [29]) and includes 343 atoms within a range of 24 A from Mg\({}^{2+}\).