| 18 Mar 2025
Variability in oxygen isotopic fractionation of enzymatic O2 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 environment- and even enzyme-level. To expand our knowledge on 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 twelve 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 to 1.034 and from 1.046 to 1.058), likely associated with the rate-limiting steps of two different O2-reduction mechanisms (sequential vs. concomitant 2-electron transfer). In comparison, iron- and copper-dependent enzymes displayed a narrower range of 18O-KIEs, with overall lower values (from 1.009 to 1.028), which increased with 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 to 1.010 and from 1.017 to 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-KIEs ranges imparted by O2-consuming enzymes and have the potential to help interpret stable O2 isotopic fractionation patterns across different environmental scales.
