What drives the variability in isotopic fractionation of O2 during enzymatic reactions?

Molecular oxygen (O₂) is one of the most important electron acceptors for a large variety of biotic and abiotic processes in the environment. A wide range of oxygen isotopic fractionation associated with biological O₂ consumption (e.g., respiration) has been reported in field and laboratory studies (¹⁸ɛ_O2 from -29 to -1 ‰). The observed variability in ¹⁸ɛ_O2 values has mainly been attributed to the different types of respiring organisms. But, to better understand what ultimately causes the variation in isotopic fractionation of O₂, it is necessary to start investigating at the lowest level of biological complexity. All biological O₂ consumption, including respiration, detoxification, and biosynthesis, occurs at the enzyme-level. A few ¹⁸ɛ_O2 values have been reported for isolated enzymatic O₂ reduction reactions. However, these laboratory-scale studies also displayed a wide range of O-isotope effects (¹⁸ɛ_O2 from -33 to -10 ‰), without any systematic correlation between ¹⁸ɛ_O2 values and the type of enzyme, substrate, or O₂-reduction mechanism. In this study, we aimed at applying O₂ stable isotope analysis to a systematic selection of O₂ consuming enzymes, to improve our molecular understanding of isotopic fractionation of O₂ at the enzyme-level. In a first series of experiments, we have determined kinetic parameters, as well as ¹⁸ɛ_O2 (and ¹⁷ɛ_O2) values of O₂ reduction for a series of copper- and flavin-dependent oxidase enzymes. O₂ reduction by these oxidase enzymes occurs separately from substrate oxidation, i.e., O₂ is reduced to water (four-electron reduction) or to hydrogen peroxide (two-electron reduction), independently from the type of substrate. Thus, the variability in observed O isotopic fractionation should only depend on the active-site structure and/or the O₂ reduction mechanism. Our experimental ¹⁸ɛ_O2 values covered the same range as those previously reported for laboratory-scale studies with other enzymes. Most of the studied flavin- and copper-dependent oxidases displayed no deviation from mass-dependent fractionation (¹⁷ɛ_O2/¹⁸ɛ_O2 ≈ 0.52). We demonstrate that ¹⁸ɛ_O2 values systematically correlate with a given enzyme’s affinity for O₂ in flavin-dependent oxidases. Furthermore, our data suggest that the range of ¹⁸ɛ_O2 and ¹⁷ɛ_O2 values differs significantly between flavin- and metal-dependent O₂ consuming enzymes. These results represent an important first step towards an improved understanding and generalization of the isotopic fractionation of O₂ at the enzyme- and, ultimately, at the organism-level.