Rubisco sustains the biosphere through the fixation of CO 2 into biomass. In plants and cyanobacteria, Form I Rubisco is structurally comprised of large and small subunits, whereas all other Rubisco Forms lack small subunits. Thus, the rise of the Form I complex through the innovation of small subunits represents a key, yet poorly understood, transition in Rubisco's evolution. Through metagenomic analyses, we discovered a previously uncharacterized clade sister to Form I Rubisco that evolved without small subunits. This clade diverged prior to the evolution of cyanobacteria and the origin of the small subunit; thus, it provides a unique reference point to advance our understanding of Form I Rubisco evolution. Structural and kinetic data presented here reveal how a proto-Form I Rubisco assembled and functioned without the structural stability imparted from small subunits. Our findings provide insight into a key evolutionary transition of the most abundant enzyme on Earth and the predominant entry point for nearly all global organic carbon.

Oligomerization is a core structural feature that defines the form and function of many proteins. Most proteins form molecular complexes; however, there remains a dearth of diversity-driven structural studies investigating the evolutionary trajectory of these assemblies. Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is one such enzyme that adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Here, we retrace the evolution of ancestral and extant form II RuBisCOs, revealing a complex and diverse history of oligomerization. We structurally characterize a newly discovered tetrameric RuBisCO, elucidating how solvent-exposed surfaces can readily adopt new interactions to interconvert or give rise to new oligomeric states. We further use these principles to engineer and demonstrate how changes in oligomerization can be mediated by relatively few mutations. Our findings yield insight into how structural plasticity may give rise to new oligomeric states.