Ribulose 1,5-bisphosphate carboxylase/oxygenase activates O₂ by electron transfer

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO₂ fixation by the biosphere. It catalyzes the addition of CO₂ onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O₂ addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O₂ is still unknown. Here, we used isotope effects (with ²H, ²⁵Mg, and ¹⁸O) to monitor O₂ activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O₂/CO₂ selectivity. Neither the Rubisco form nor the use of solvent D₂O and deuterated RuBP changed the ¹⁶O/¹⁸O isotope effect of O₂ addition, in clear contrast with the ¹²C/¹³C isotope effect of CO₂ addition. Furthermore, substitution of light magnesium (²⁴Mg) by heavy, nuclear magnetic ²⁵Mg had no effect on O₂ addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O₂) does not seem to be involved in O₂ activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O₂^•-) production, must be insensitive to deuteration, and yields a predicted ¹⁶O/¹⁸O isotope effect and energy barrier close to observed values. Overall, O₂ undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.