Crystallographic, mutagenesis, kinetic, and computational studies on Rubisco over three decades have revealed much about its catalytic mechanism and the role played by several active-site residues. However, key questions remain unanswered. Specific details of the carboxylase and oxygenase mechanisms, required to underpin the rational re-engineering of Rubisco, are still speculative. Here we address critical gaps in knowledge with a definitive comprehensive computational investigation of the mechanism of carboxylase activity at the Rubisco active site. Density functional theory calculations (B3LYP/6-31G(d,p)) were performed on active-site fragment models of a size up to 77 atoms, not previously possible computationally. All amino acid residues suspected to play roles in the acid-base chemistry in the multistep reaction, and interacting directly with the central Mg (2+) atom and the reactive moiety of substrate and intermediates, were included. The results provide a firm basis for us to propose a novel mechanism for the entire sequence of reactions in the carboxylase catalysis and to define precise roles for the active-site residues, singly and in concert. In this mechanism, the carbamylated LYS201 plays a more limited role than previously proposed but is crucial for initiating the reaction by acting as a base in the enolization. We suggest a wider role for HIS294, with involvement in the carboxylation, hydration, and C2-C3 bond-scission steps, consistent with the suggestion of Harpel et al. (1998) but contrary to the consensus view of Cleland et al. (1998). In contrast to the common assumption that the water molecule for the hydration step comes from within the active site, we propose that the Mg-coordinated water is not dissociated at the start of the gas-addition reaction but rather remains coordinated and is used for the hydration of the C3 carbon atom. New roles are also proposed for LYS175, GLU204, and HIS294. The mechanism suggests roles in the gas-addition step for residues in three spatially distinct regions of the active site, HIS294 and LYS334 in the C-terminal domain of the large subunit (LSU), but also hitherto unsuspected roles for a cluster of three residues (ASN123, GLU60, and TYR20) in the N-terminal domain of the partner LSU of the dimer containing the active site. Our new mechanism is supported by existing experimental data, provides new convincing interpretations of previously puzzling data, and allows new insights into mutational strategies for improving Rubisco activity.

The ubiquitous enzyme Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) fixes atmospheric carbon dioxide within the Calvin-Benson cycle that is utilized by most photosynthetic organisms. Despite this central role, RuBisCO's efficiency surprisingly struggles, with both a very slow turnover rate to products and also impaired substrate specificity, features that have long been an enigma as it would be assumed that its efficiency was under strong evolutionary pressure. RuBisCO's substrate specificity is compromised as it catalyzes a side-fixation reaction with atmospheric oxygen; empirical kinetic results show a trend to tradeoff between relative specificity and low catalytic turnover rate. Although the dominant hypothesis has been that the active-site chemistry constrains the enzyme's evolution, a more recent study on RuBisCO stability and adaptability has implicated competing selection pressures. Elucidating these constraints is crucial for directing future research on improving photosynthesis, as the current literature casts doubt on the potential effectiveness of site-directed mutagenesis to improve RuBisCO's efficiency. Here we use regression analysis to quantify the relationships between kinetic parameters obtained from empirical data sets spanning a wide evolutionary range of RuBisCOs. Most significantly we found that the rate constant for dissociation of CO2 from the enzyme complex was much higher than previous estimates and comparable with the corresponding catalytic rate constant. Observed trends between relative specificity and turnover rate can be expressed as the product of negative and positive correlation factors. This provides an explanation in simple kinetic terms of both the natural variation of relative specificity as well as that obtained by reported site-directed mutagenesis results. We demonstrate that the kinetic behaviour shows a lesser rather than more constrained RuBisCO, consistent with growing empirical evidence of higher variability in relative specificity. In summary our analysis supports an explanation for the origin of the tradeoff between specificity and turnover as due to competition between protein stability and activity, rather than constraints between rate constants imposed by the underlying chemistry. Our analysis suggests that simultaneous improvement in both specificity and turnover rate of RuBisCO is possible.

Here, we describe a computational approach for studying enzymes that catalyze complex multi-step reactions and apply it to Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5-step carboxylase reaction, the substrate Ribulose-1,5-bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO . Hydration of the RuBP.CO complex is followed by CC bond scission and stereospecific protonation. However, details of the roles and protonation states of active-site residues, and sources of protons and water, remain highly speculative. Large-scale computations on active-site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi-empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO complex and associated active-site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO ; both hydration and CC bond scission require early protonation of CO in the RuBP.CO complex; CC bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3-gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis-Menten-like complex corresponding to the empirical K (=K ) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.

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.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the primary carbon-fixing enzyme in photosynthesis, fixing CO2 to a 5-carbon sugar, RuBP, in a series of five reactions. However, it also catalyzes an oxygenase reaction by O2 addition to the same enolized RuBP substrate in an analogous reaction series in the same active site, producing a waste product and loss of photosynthetic efficiency. Starting from RuBP, the reactions are enolization to the enediolate form, addition of CO2 or O2 to form the carboxy or peroxo adduct, hydration to form a gemdiolate, scission of the C2–C3 bond of the original RuBP, and stereospecific or nonstereospecific protonation to form two molecules of the 3-carbon PGA product, or one molecule of PGA, one of 2-carbon PG (waste product), and one water molecule. Reducing the loss of efficiency from the oxygenase reaction is an attractive means to increase crop productivity. However, lack of understanding of key aspects of the catalytic mechanisms for both the carboxylase and oxygenase reactions, particularly those involving proton exchanges and roles of water molecules, has stymied efforts at re-engineering Rubisco to reduce losses from the oxygenation reaction. As the stable form of molecular oxygen is the triplet biradical state (3O2), its reaction with near-universal singlet-state molecules is formally spin forbidden. Although in oxygenase enzymes, 3O2 activation is usually achieved by one-electron transfers using transition-metal ions or organic cofactors, recently, cofactor-less oxygenases in which the substrate itself is the source of the electron for 3O2 activation have been identified, but in all such cases an aromatic ring stabilizes the substrate’s negative charge. Here we present the first large-scale Kohn–Sham density functional theory study of the reaction mechanism of the Rubisco oxygenase pathway. First, we show that the enediolate substrate complexed to Mg2+ and its ligands extends the region for charge delocalization and stabilization of its negative charge to allow formation of a caged biradical enediolate–O2 complex. Thus, Rubisco is a unique type of oxygenase without precedent in the literature. Second, for the O2 addition to proceed to the singlet peroxo-adduct intermediate, the system must undergo an intersystem crossing. We found that the presence of protonated LYS334 is required to stabilize this intermediate and that both factors (strongly stabilized anion and protonated LYS334) facilitate a barrier-less activation of 3O2. This finding supports our recent proposal that deoxygenation, that is, reversal of gas binding, is possible. Third, as neither CO2 nor O2 binds to the enzyme, our findings support the proposal from our recent carboxylase study that the observed K C or K O (Michaelis–Menten constants) in the steady-state kinetics reflect the respective adducts, carboxy or peroxo. Fourth, after computing hydration pathways with water addition both syn and anti to C3, we found, in contrast to the results of our carboxylation study indicating anti addition, ...