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

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO fixation by the biosphere. It catalyzes the addition of CO 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 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 is still unknown. Here, we used isotope effects (with H, Mg, and O) to monitor O activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O/CO selectivity. Neither the Rubisco form nor the use of solvent DO and deuterated RuBP changed the O/O isotope effect of O addition, in clear contrast with the C/C isotope effect of CO addition. Furthermore, substitution of light magnesium (Mg) by heavy, nuclear magnetic Mg had no effect on O addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O) does not seem to be involved in O activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O) production, must be insensitive to deuteration, and yields a predicted O/O isotope effect and energy barrier close to observed values. Overall, O undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate.