Rubisco proton production can drive the elevation of CO ₂ within condensates and carboxysomes

Abstract

Membraneless organelles containing the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are a common feature of organisms utilizing CO2 concentrating mechanisms to enhance photosynthetic carbon acquisition. In cyanobacteria and proteobacteria, the Rubisco condensate is encapsulated in a proteinaceous shell, collectively termed a carboxysome, while some algae and hornworts have evolved Rubisco condensates known as pyrenoids. In both cases, CO2 fixation is enhanced compared with the free enzyme. Previous mathematical models have attributed the improved function of carboxysomes to the generation of elevated CO2 within the organelle via a colocalized carbonic anhydrase (CA) and inwardly diffusing HCO3−, which have accumulated in the cytoplasm via dedicated transporters. Here, we present a concept in which we consider the net of two protons produced in every Rubisco carboxylase reaction. We evaluate this in a reaction–diffusion compartment model to investigate functional advantages these protons may provide Rubisco condensates and carboxysomes, prior to the evolution of HCO3− accumulation. Our model highlights that diffusional resistance to reaction species within a condensate allows Rubisco-derived protons to drive the conversion of HCO3− to CO2 via colocalized CA, enhancing both condensate [CO2] and Rubisco rate. Protonation of Rubisco substrate (RuBP) and product (phosphoglycerate) plays an important role in modulating internal pH and CO2 generation. Application of the model to putative evolutionary ancestors, prior to contemporary cellular HCO3− accumulation, revealed photosynthetic enhancements along a logical sequence of advancements, via Rubisco condensation, to fully formed carboxysomes. Our model suggests that evolution of Rubisco condensation could be favored under low CO2 and low light environments.

Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time

“…It has been theorized that RuBisCO kinetics have adapted in response to CO2 availability, either due to increased environmental CO2 (36) or the emergence of CCMs (e.g., C4 photosynthesis in plants ( 60)). Considering the positive relationship between enzymatic fractionation and RuBisCO's specificity to CO2 (36), reconstructing ancient RuBisCO kinetic isotope effect could provide insights into the co-evolution of atmospheric concentrations of CO2 and O2 and carbon fixation strategies during the Precambrian, in particular the emergence of carbon concentrating mechanisms (CCMs) (34,(61)(62)(63). This is relevant as precise estimates of the magnitude of atmospheric CO2 elevation during the Precambrian relative to the present, as well as the emergence and effectiveness of Precambrian CCMs, are unknown.…”

“…These differences are contingent upon changes to internal (cellular, physiological) and external (environmental) conditions that have demonstrably varied over Earth's long history. Cyanobacteria, with well-characterized genetic and morphological features (40,41,(61)(62)(63) and a tractable paleobiological history (18,68), are ideal hosts for investigating a range of early Precambrian metabolic processes (68)(69)(70). Discernible trends (or steadfast consistencies) in metabolic outputs over macroevolutionary timescales can lead to foundational uniformitarian approaches to deep time molecular paleobiology.…”

Engineered Accumulation of Bicarbonate in Plant Chloroplasts: Known Knowns and Known Unknowns

“…The efficiency of Rubisco carboxylation is hampered by O 2 , leading to photorespiratory expenditure of accumulated CO 2 and chemical energy ( Busch, 2020 ). It is assumed that factors selecting for maintenance of relatively high rates of carboxylation, as atmospheric concentrations of CO 2 decreased while O 2 increased approximately 350 million years ago, may have led to a divergence in mechanistic adaptations between aquatic and terrestrial photosynthetic organisms ( Flamholz and Shih, 2020 ; Long et al, 2021 ). Thus, cyanobacteria and many eukaryotic algae evolved CCMs to overcome these challenges, while emerging terrestrial C 3 plants have maintained a larger investment in Rubisco and evolved to maximize beneficial biochemical contributions from photorespiratory nitrogen and sulfur metabolism ( Shi and Bloom, 2021 ).…”

Computational Modeling and Evolutionary Implications of Biochemical Reactions in Bacterial Microcompartments

“…They determined the importance of HCO3and pH on CO2 fixation rate and that elevated pH impacted CO2 fixation through processes outside of the CCM [44]. Most recently, Long et al modeled the inclusion of 'proton transport' in and out of the carboxysome via RuBP and PGA, the substrate and product of RuBisCO's carboxylase activity respectively [45]. This paper placed the carboxysome in a m 3 cytosolic solution and excluded the HCO3transport aspect of the CCM (Table 1).…”