Mechanism of Oxygenase-Pathway Reactions Catalyzed by Rubisco from Large-Scale Kohn–Sham Density Functional Calculations

Abstract

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, ...

Response: Commentary: Directions for Optimization of Photosynthetic Carbon Fixation: RuBisCO’s Efficiency May Not Be So Constrained After All

“…It is possible that both binding and dissociation are feasible by flipping between singlet and triplet states where the energy surfaces crossover (intersystem crossing). This has been verified by our quantum chemical calculations (Kannappan et al, 2019). Note that this is the first study in the literature that has addressed directly how the formally spin-forbidden oxygenation step (triplet O 2 to singlet peroxo adduct) can be achieved.…”

“…Point 1: Our recent computational study (Kannappan et al, 2019) suggests that rates of catalysis and deoxygenation may well be very similar. The overall reaction, i.e., to products, is certainly highly exothermic, so it clearly cannot be reversed.…”

The Coevolution of RuBisCO, Photorespiration, and Carbon Concentrating Mechanisms in Higher Plants

“…This tends to suggest some level of correlation between carboxylase and oxygenase k i , most likely with those associated with substrate affinity ( Figure 6 ). Both substrates present similar electrostatic potentials to the RuBisCO active site ( Kannappan and Gready, 2008 ), so that the binding of CO 2 to enolized RuBP induces a redistribution of charge similar to that induced by O 2 binding ( Cummins et al, 2018b ; Kannappan et al, 2019 ; Bathellier et al, 2020 ; Cummins and Gready, 2020 ) which will then interact similarly with the external (to the active site) electrostatic field, which is thought to be the primary driver in enzyme catalysis ( Warshel et al, 2006 ; Fried and Boxer, 2017 ). Moreover, evolutionary changes in this electrostatic field have been linked to RuBisCO substrate specificity ( Poudel et al, 2020 ) This electrostatic field would change slightly with sequence variation outside the highly conserved active site to produce the small free energy changes required to maintain the correlation between S C and S O when mutations occur.…”

“…The combined effects of increasing population and anthropogenic climate change have motivated efforts to enhance carbon fixation in plants for increasing both agricultural crop yield and carbon sequestration generally ( Niinemets et al, 2017 ; Andralojc et al, 2018 ; Erb and Zarzycki, 2018 ; Fernie et al, 2020 ; Lawson and Flexus, 2020 ). Although the possibility of enhancing photosynthesis by improving RuBisCO kinetic traits has been given due consideration ( Whitney et al, 2011 ; Sharwood et al, 2016 ; Gomez-Fernandez et al, 2018 ; Wilson et al, 2018 ; Zhou and Whitney, 2019 ; Davidi et al, 2020 ; Lin et al, 2020 ; Bouvier et al, 2021 ), a conclusive picture of RuBisCO’s molecular mechanism ( Cleland et al, 1998 ; Tcherkez, 2013 , 2015 ; Cummins et al, 2018b , 2019b ; Kannappan et al, 2019 ; Bathellier et al, 2020 ; Cummins and Gready, 2020 ) and a general consensus understanding of the observed tradeoffs between RuBisCO’s kinetic parameters remains elusive, despite having been analyzed in varying ways with the objective of gaining insights into the possible connection between evolutionary and biochemical (or catalytic) constraints ( Bouvier et al, 2021 ). The earliest of these studies, ( Tcherkez et al, 2006 ; Savir et al, 2010 ) based on general mechanistic assumptions and limited data samples, concluded that variations in the elementary rate constants must be tightly constrained by the limitations inherent in RuBisCOs kinetic mechanism, resulting in an enzyme which provides only limited scope for further optimization ( Tcherkez et al, 2006 ; Tcherkez, 2013 , 2015 ), while later studies of more extensive data sets have challenged this view, revealing greater flexibility ( Cummins et al, 2018a , 2019a ; Flamholz et al, 2019 ).…”

Ribulose 1,5-bisphosphate carboxylase/oxygenase activates O 2 by electron transfer

“…However, H 2 O 2 has been shown to be generated during oxygenation (about one molecule per 200 catalytic cycles) (18), suggesting that a reactive oxygen species might be involved in the mechanism (or that the peroxide can be broken down, yielding hydrogen peroxide). Ab initio calculations with density functional theory (DFT) using energy expression based on hybrid energy functionals B3LYP have suggested that SET is plausible, since the minimum-energy structure with bound oxygen is found to correspond to a biradical complex with partial electron transfer to O 2 (19). It has been suggested that most cofactor-less enzymes such as glucose oxidase activate O 2 by SET followed by spin transition via spin orbit coupling (SOC) (14,20), and Rubisco, perhaps, involves a similar mechanism.…”