Atomic resolution structure of spinach rubisco reveals protons and dynamics

Abstract

Photosynthetic organisms sustain life on Earth by storing solar energy in biomass. Central to this process is rubisco, the enzyme that catalyses the fixation of CO2 to ribulose-1,5-bisphosphate, providing the primary gateway for inorganic carbon into the biosphere. Rubiscos catalytic efficiency is a major determinant of crop productivity and global carbon flux, making it a longstanding target for protein engineering.1-4 Yet, attempts to enhance its performance through rational design have met limited success due to an incomplete understanding of rubiscos catalytic mechanism. Here, we report an atomic resolution (1.25 [A]) cryo-EM structure of spinach rubisco in complex with the transition-state analogue 2-carboxyarabinitol-1,5-bisphosphate. Supported by large-scale quantum/classical (QM/MM) calculations, our structural analysis reveals protonation equilibria within the active site and unexpected structural flexibility across large protein regions despite the exceptionally tight ligand binding. Our findings provide new insight into the complex interplay of protonation equilibria and conformational sampling, suggesting a novel basis for rubiscos rational redesign utilizing strategies that rely on a combination of dynamic and electrostatic control.