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 O-2 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 O-2 is still unknown. Here, we used isotope effects (with H-2, Mg-25, and O-18) to monitor O-2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O-2/CO2 selectivity. Neither the Rubisco form nor the use of solvent D2O and deuterated RuBP changed the O-16/O-18 isotope effect of O-2 addition, in clear contrast with the C-12/C-13 isotope effect of CO2 addition. Furthermore, substitution of light magnesium (Mg-24) by heavy, nuclear magnetic Mg-25 had no effect on O-2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O-2) does not seem to be involved in O-2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O-2(center dot-)) production, must be insensitive to deuteration, and yields a predicted O-16/O-18 isotope effect and energy barrier close to observed values. Overall, O-2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.