Variability in oxygen isotopic fractionation of enzymatic O ₂ consumption

Abstract. Stable isotope analysis of O2 has emerged as a valuable tool to study O2 dynamics at various environmental scales, from molecular mechanisms to ecosystem processes. Despite its utility, there is a lack of fundamental understanding of the large variability observed in O2 isotopic fractionation at the environmental and even enzymatic levels. To expand our knowledge of the potential causes of this variability, we determined 18O kinetic isotope effects (KIEs) across a broad range of O2-consuming enzymes. The studied enzymes included nine flavin-dependent, five copper-dependent, and one copper-heme-dependent oxidases, as well as one flavin-dependent monooxygenase. For 12 of these enzymes, 18O KIEs were determined for the first time. The comparison of 18O KIEs, determined in this and previous studies, to calculated 18O equilibrium isotope effects revealed distinct patterns of O isotopic fractionation within and between enzyme groups, reflecting differences in active-site structures and O2-reduction mechanisms. Flavin-dependent O2-consuming enzymes exhibited two distinct ranges of 18O KIEs (from 1.020–1.034 and from 1.046–1.058), likely associated with the rate-limiting steps of two different O2-reduction mechanisms (sequential vs. concomitant two-electron transfer). In comparison, iron- and copper-dependent enzymes displayed a narrower range of 18O KIEs, with overall lower values (from 1.009–1.028), associated with an increase in the degree of O2 reduction during the rate-limiting step. Similar to flavin-dependent O2-consuming enzymes, copper-dependent O2-consuming enzymes also featured two main, yet narrower, ranges of 18O KIEs (from 1.009–1.010 and from 1.017–1.022), likely associated with the rate-limiting formation of a copper-superoxo or copper-hydroperoxo intermediate. Overall, our findings support generalizations regarding expected 18O KIE ranges imparted by O2-consuming enzymes and have the potential to help interpret stable O2 isotopic fractionation patterns across different environmental scales.