## 5 CO\({}_{2}\)/O\({}_{2}\) Molecular Recognition Strategy
(Figure from ref. [15], used by permission of PNAS (copyright © 2012)). vated in a dodecahedron cell with an edge length of 12 nm and the desired amount of gas molecules was created to model the CO\({}_{2}\) and O\({}_{2}\) migration around the protein. The L\({}_{6}\)S\({}_{8}\) models reproduced the protein structure of Rubisco from spinach (PDB access code 1 aus, 1 ruc and 1 rcx) and from _Chlamidomonas reinhardtii_ (PDB access code 1 gk8). On the basis of the partition of the two gases between the protein and the surrounding solution, the authors were able to calculate the intrinsic affinity of Rubisco for CO\({}_{2}\) as +0.3 kJ/mol compared to +10 kJ/mol calculated for O\({}_{2}\). These data indicate that the whole protein binds CO\({}_{2}\) stronger that O\({}_{2}\). In agreement with [PERSON], the authors support the stronger CO\({}_{2}\) protein-binding with the difference in molecular quadrupoles of the two ligands. Moreover, by modelling the CO\({}_{2}\) and O\({}_{2}\) densities around the active site of Rubisco from _Clamidomonas reinhardtii_, the authors found that CO\({}_{2}\) has a preference for binding a region around the active site that extends to the closest S subunit (Figure S4). The simulation showed that the overall S subunit has a higher affinity for CO\({}_{2}\) than the overall L subunit suggesting that the S subunit plays a role as \"CO\({}_{2}\) reservoir\" for catalysis. The hypothesis advanced by [PERSON] is supported to some degree by a recent study by [PERSON] et al. [32] which analyzed the high resolution crystal structures of 11 Rubisco proteins stored in the PDB. The structural study was integrated with a phylogenetic analysis constructed using 55 Rubisco proteins sequences that confirmed that Rubisco homologs are assigned into four different phyletic groups named G-I, G-II, GIII and G-IV (the four forms are usually designated as form I, II, III and IV as mentioned in Section 3). Moreover the study demonstrated that: (i) G-II enzymes evolved from proteobacteria; (ii) G-III Rubisco evolved from the archaeal domain; (iii) G-I Rubisco evolved later being represented among bacteria, eukaryotic algae and higher plants. Distinct subclass of Rubisco proteins were found among the G-I group (i.e., IA, IB, IC and ID) showing a wide range of S\({}_{\text{C/O}}\) values ranging from 40 to 110. The 11 high resolution crystal structures belonging to the monophyletic groups G-I and G-II allowed the observation that amino acid residues proximal to the active sites are not strictly conserved among different homologs and are likely sites for evolution. Crystal structures assigned the PBD ID5 MAC, 1 BXN and 4 FOK are related to G-II, G-IC and G-ID Rubisco respectively, and belong to proteins progressively adapted to increasing O\({}_{2}\) concentration. Interestingly, comparison of these crystal structures evidenced the presence of higher surface charge potential proximal to the active site and solvent accessible channels connecting the protein surface to the active site. Enzymes bearing these larger positive channels easily \"drive\" CO\({}_{2}\) towards the active site, thanks to the electrostatic steering with the CO\({}_{2}\) quadrupole coming from a partial negative charge on the two oxygen atoms. Most important is that the extension of the positive charged areas progressively increases from 5 MAC to 1 BXN structures reaching the maximum extension in the 4 FOK structure. Interestingly, increasing extension of the positive charged areas within the three proteins parallels increased S\({}_{\text{C/O}}\) selectivity.