Variability in oxygen isotopic fractionation of enzymatic O2 consumption

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 copperdependent
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 ratelimiting
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 copperhydroperoxo
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.