What Fractionates Oxygen Isotopes during Respiration? Insights from Multiple Isotopologue Measurements and Theory

The precise mass dependence of respiratory O2 consumption
underpins the “oxygen triple-isotope” approach to quantifying
gross primary productivity in modern and ancient environments. Yet,
the physical-chemical origins of the key 18O/16O and 17O/16O covariations observed during
respiration have not been tied to theory; thus, the approach remains
empirical. First-principles calculations on enzyme active-site models
suggest that changes in the O–O bond strength upon electron
transfer strongly influence respiratory isotopic fractionation. However,
molecular diffusion may also be important. Here, we use measurements
of the relative abundances of rare isotopologues 17O18O and 18O18O as additional tracers
of mass dependence during dark respiration experiments of lacustrine
water. We then compare the experimental results to first-principles
calculations of O2 interacting with heme-oxidase analogues.
We find a significantly steeper mass dependence, supported by theory,
than has been previously observed. Enrichments of 17O18O and 18O18O in the O2 residue
suggest that θ values are strongly influenced by chemical processes,
rather than being dominated by physical processes (i.e., by bond alteration
rather than diffusion). In contrast, earlier data are inconsistent
with theory, implying that analytical artifacts may have biased those
results. Implications for quantifying primary productivity are discussed.