CO2 fixation by Rubisco: Computational dissection of the key steps of carboxylation, hydration, and C-C bond cleavage

Despite intensive experimental and computational studies, some important fe atures of the mechanism of the photosynthetic CO2-fixing enzyme, Rubisco, a re still not understood. To complement our previous investigation of the fi rst catalytic step, the enolization Of D-ribulose-1,5-bisphosphate (King et al., Biochemistry 1998, 44, 15414-15422), we present the first complete co mputational dissection of subsequent steps of the carboxylation reaction th at includes the roles of the central magnesium ion and modeled residues of the active site. We investigated carboxylation, hydration, and C-C bond cle avage using the density functional method and the B3LYP/6-31G(d) level to p erform geometry optimizations. The energies were determined by B3LYP/6-311G(2d,p) single-point calculations. We modeled a fragment of the active site and substrate, taking into account experimental findings that the residues coordinated to the Mg ion, especially the carbamylated Lys-201, play criti cal roles in this reaction sequence. The carbamate appears to act as a gene ral base, not only for enolization but also for hydration of the ketoacid b eta formed by addition of CO2 and, as well, cleavage of the C2-C3 bond of t he hydrate. We show that CO2 is added directly, without assistance of a Mic haelis complex, and that hydration of the resultant beta ketoacid occurs in a separate subsequent step with a discrete transition state. We suggest th at two conformations of the hydrate (gem-diol), with different metal coordi nation, are possible. The step with the highest activation energy during th e carboxylation cycle is the C-C bond cleavage. Depending on the conformati ons of the gem-diol, different pathways are possible for this step. In eith er case, special arrangements of the metal coordination result in bond brea king occurring at remarkably low activation energies (between 28 and 37 kca l mol(-1)) which might be reduced further in the enzyme environment.