Ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms from diverse species show differences in oligomeric structure, thermal stability, and activity.

Rubisco activase (Rca) is critical for maintaining Rubisco activity during photosynthesis by removing inhibitory sugar phosphates through ATP hydrolysis. Despite its importance, the structural and functional diversity of Rca across species remain poorly understood. This study compares the oligomeric assembly, thermal stability, and functional activities of α- and β-isoforms of Rca from cotton, creosote, Antarctic hairgrass, and Sitka spruce, representing diverse thermal and ecological adaptations. We found that cotton and creosote Rca isoforms form highly polydisperse complexes in solution, with no evidence of discrete hexamer formation, even in the presence of Mg.ATPγS. In contrast, Antarctic hairgrass α-Rca and Sitka spruce β-Rca formed stable hexamers under similar conditions. Spruce α-Rca exhibited unique redox-dependent oligomerization, forming large complexes stabilized by disulfide bonds. Thermal stability assays revealed significant nucleotide-induced stabilization in most isoforms, with hexamer formation enhancing stability and activity in select cases. Functional assays showed that hexamer-forming isoforms displayed superior Rubisco reactivation and ATP hydrolysis activities, even at low protein concentrations, while smaller oligomeric assemblies also supported activity in some species. These findings provide new insights into the structural and functional adaptations of Rca, highlighting the role of oligomeric assembly and environmental influences on its activity. This work lays a foundation for improving photosynthetic efficiency by targeting Rca isoforms tailored to specific environmental conditions.