Up to now, the evolutionary trajectory of the most prevalent CO2-fixing enzyme – Rubisco Form I, has been difficult to study. The Rubisco found in today’s plants and algae has 8 small subunits (SSU) that are absent from its ancestral form. The SSU has become essential for the enzyme’s solubility and activity, hindering biochemical studies into its influence on Rubisco. Our research aimed to decipher how the obligate interaction between Rubisco and its SSU evolved. This includes identifying the functional implications of Rubisco interacting with the SSU, discovering why the modern Rubisco depends on the SSU, as well as how the SSU modulated the evolutionary trajectories available to Rubisco. To address these questions, we combined ancestral sequence reconstruction and mass photometry. We re-created a long-extinct evolutionary precursor of modern Rubiscos, which interacts with the SSU but does not depend on it. With this intermediate, we show that Rubisco increased its specificity and carboxylation efficiency through the gain of a new accessory subunit before atmospheric oxygen was present.

Mass photometry enabled us to narrow down the evolutionary interval over which Rubisco’s oligomerization state changed. It also made it possible to screen for the substitutions that caused Rubisco’s dependence on the SSU for solubility.

We show that the current form of Rubisco evolved from an octamer of large subunits that started to interact with the SSU more than 2.5 billion years ago. Immediately upon recruitment, the SSU improved Rubiscos catalysis and specificity. Then, a substitution made Rubisco dependent on the SSU for solubility. Our work sheds light on the emergence of an adaptation to rising ambient oxygen levels, provides a template for investigating the function of interactions that have remained elusive because of their essentiality, and sheds new light on the determinants of specificity in Rubisco.

 

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