Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the primary carbon-fixing enzyme in photosynthesis, fixing CO2 to a 5-carbon sugar, RuBP, in a series of five reactions. However, it also catalyzes an oxygenase reaction by O2 addition to the same enolized RuBP substrate in an analogous reaction series in the same active site, producing a waste product and loss of photosynthetic efficiency. Starting from RuBP, the reactions are enolization to the enediolate form, addition of CO2 or O2 to form the carboxy or peroxo adduct, hydration to form a gemdiolate, scission of the C2-C3 bond of the original RuBP, and stereospecific or nonstereospecific protonation to form two molecules of the 3-carbon PGA product, or one molecule of PGA, one of 2-carbon PG (waste product), and one water molecule. Reducing the loss of efficiency from the oxygenase reaction is an attractive means to increase crop productivity. However, lack of understanding of key aspects of the catalytic mechanisms for both the carboxylase and oxygenase reactions, particularly those involving proton exchanges and roles of water molecules, has stymied efforts at re-engineering Rubisco to reduce losses from the oxygenation reaction. As the stable form of molecular oxygen is the triplet biradical state (3O2), its reaction with near-universal singlet-state molecules is formally spin forbidden. Although in oxygenase enzymes, 3O2 activation is usually achieved by one-electron transfers using transition-metal ions or organic cofactors, recently, cofactor-less oxygenases in which the substrate itself is the source of the electron for 3O2 activation have been identified, but in all such cases an aromatic ring stabilizes the substrate's negative charge. Here we present the first large-scale Kohn-Sham density functional theory study of the reaction mechanism of the Rubisco oxygenase pathway. First, we show that the enediolate substrate complexed to Mg2+ and its ligands extends the region for charge delocalization and stabilization of its negative charge to allow formation of a caged biradical enediolate-O2 complex. Thus, Rubisco is a unique type of oxygenase without precedent in the literature. Second, for the O2 addition to proceed to the singlet peroxo-adduct intermediate, the system must undergo an intersystem crossing. We found that the presence of protonated LYS334 is required to stabilize this intermediate and that both factors (strongly stabilized anion and protonated LYS334) facilitate a barrier-less activation of 3O2. This finding supports our recent proposal that deoxygenation, that is, reversal of gas binding, is possible. Third, as neither CO2 nor O2 binds to the enzyme, our findings support the proposal from our recent carboxylase study that the observed KC or KO (Michaelis-Menten constants) in the steady-state kinetics reflect the respective adducts, carboxy or peroxo. Fourth, after computing hydration pathways with water addition both syn and anti to C3, we found, in contrast to the results of our carboxylation study indicating anti addition, that in the oxygenation reaction only syn-hydration is capable of producing a stable gemdiolate that facilitates the rate-limiting C2-C3 bond scission to final products. Fifth, we propose that an excess proton we previously found was required in the carboxylation reaction for activating the C2-C3 bond scission is utilized in the oxygenation reaction for the required elimination of a water molecule. In summary, despite its oxygenase handicap, Rubisco's success in directing 75% of its substrate through the carboxylation pathway can be considered impressively effective. Although native C3 Rubiscos are in a fix with unwanted activity of 3O2 hampering its primary carboxylase function, mechanistic differences presented here with findings in our recent carboxylase study for both the gas-addition and subsequent reactions provide some clues as to how creative Rubisco re-engineering may offer a solution to reducing the oxygenase activity.