Ab Initio Molecular Dynamics Simulation and Energetics of the Ribulose-1,5-biphosphate Carboxylation Reaction Catalyzed by Rubisco: Toward Elucidating the Stereospecific Protonation Mechanism

Abstract

In the carboxylation reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) carboxylase–oxygenase (Rubisco), which is fundamental to photosynthesis, scission of a C–C bond in the six-carbon gemdiolate intermediate forms a carbanion that must be protonated stereospecifically to form product. It is thought that a conserved lysine side chain (LYS175 in spinach Rubisco), in the immediate vicinity of the carbanion, provides the necessary proton. Here, we endeavor to determine from the electronic-structure calculations whether protonation via this route is energetically possible. The two-dimensional energy surface was mapped to determine the minimum energy path (MEP) using density functional theory (B3LYP) and incorporating basis set superposition and classical (London) dispersion corrections. The potential of mean force (free energy) was then calculated from ab initio molecular dynamics simulations with umbrella sampling in the vicinity of the MEP on the scission–protonation reaction coordinate. MEP calculations were also carried out to evaluate the possibility of an active-site water near the phosphate (P1) of RuBP, with an excess proton positioned at P1, as an alternative facilitator of stereospecific protonation via a classical Grotthuss mechanism. In both cases, the C–C bond scission in the six-carbon intermediate and proton transfer from the donor was found to be concerted and highly asynchronous, without a stable carbanion intermediate. However, the free energy change was unfavorable for direct protonation by the LYS175 side chain. In contrast, the Grotthuss mechanism yielded stable products and an activation energy in good agreement with experiment. It also provides a plausible mechanism for alternative product formed in enzyme mutations at the LYS175 position and is consistent with the observed deuterium isotope effects.

Electronic Structure Benchmark Calculations of CO2 Fixing Elementary Chemical Steps in RuBisCO Using the Projector-Based Embedding Approach

“…1,2,[4][5][6][7][8][9] Within the enzyme's catalytic pocket, a set of five chemical reactions convert the initial substrate, ribulose 1,5-bisphosphate (RuBP), to the final product, two-molecules of 3-phosphoglycerate (3-PGA). [4][5][6]10,11 The catalyzed chemical reactions involve CO 2 and H 2 O addition to an activated RuBP intermediate by enolization, which subsequently undergoes a bond dissociation reaction to produce two-molecules of 3-PGA through stereospecific protonation. 5,6,[10][11][12] All these chemical transformations (enolization, carboxylation, hydration, bond cleavage and stereospecific protonation) occur in a highly charged chemical environment centered on the first magnesium (II) coordination sphere (see Figure 1A).…”

“…[4][5][6]10,11 The catalyzed chemical reactions involve CO 2 and H 2 O addition to an activated RuBP intermediate by enolization, which subsequently undergoes a bond dissociation reaction to produce two-molecules of 3-PGA through stereospecific protonation. 5,6,[10][11][12] All these chemical transformations (enolization, carboxylation, hydration, bond cleavage and stereospecific protonation) occur in a highly charged chemical environment centered on the first magnesium (II) coordination sphere (see Figure 1A). 4,5 In the last five decades, much effort has been devoted to studying the mechanisms of RuBisCO carboxylase activity.…”

Electronic structure benchmark calculations of CO2 fixing elementary chemical steps in RuBisCO using the projector‐based embedding approach

“…[ 1,2,4–9 ] Within the enzyme's catalytic pocket, a set of five chemical reactions convert the initial substrate, ribulose 1,5‐bisphosphate (RuBP), to the final product, two‐molecules of 3‐phosphoglycerate (3‐PGA). [ 4–6,10,11 ] The catalyzed chemical reactions involve CO 2 and H 2 O addition to an activated RuBP intermediate by enolization, which subsequently undergoes a bond dissociation reaction to produce two‐molecules of 3‐PGA through stereospecific protonation. [ 5,6,10–12 ] All these chemical transformations (enolization, carboxylation, hydration, bond cleavage, and stereospecific protonation) occur in a highly charged chemical environment centered on the first magnesium (II) coordination sphere (see Figure 1a).…”

“…[ 5,6,10–14 ] Computational and experimental studies have identified the role of different amino‐acid residues within each chemical step. [ 4–7,10–24 ] Specifically, Gready et al [ 5 ] have proposed a complete sequence of chemical species that connect the initial RuBP with the two‐molecules of 3‐PGA from calculations on a set of model fragments representing the active‐site and substrate. These models represent the first magnesium coordination sphere including a reduced version of the substrate and the protonated residues Kcx201 (carbamylated lysine), Asp203 and Glu204 together with the second magnesium coordination sphere consisting of residues His294, Lys334, Lys177, and Lys175, schematically shown in Figure 1a.…”

“…Mechanistic details are still debated and the focus of ongoing research on RuBisCO. [ 6,10–14 ] The proposed reaction path by Gready et al still represents the largest and most complete model reported so far to describe all chemical events that take place within enzyme active‐site. However, a systematic study of the effect of the electronic structure method used to describe the energetics associated with each chemical step (activation and reaction energy) is still missing.…”