Comments by the referee are marked as “Ref {comment no.}”, answers by the authors are given as “Auth {comment no.}”. The text extracts following author comments show the revised parts of the manuscript with line numbers corresponding to the original submission.

Ref 1.0 The manuscript describes the isotope effects of O2-consuming enzymes and will be useful for many disciplines that seek to investigate respiration processes in the environment. The authors have tested a wide range of organic substrates and corresponding enzymes, extending the dataset in the literature, and have provided useful conjecture on mechanisms that may determine isotope distributions. The isotope methodology is modern, eloquent, and explained in detail. I lack the expertise to provide a thorough review of the experimental set-up and concentrations chosen to perform the enzyme assays; however, the text explaining these choices is transparent and polished, and for my understanding, sufficient information has been provided in the Appendices and available data sets. I recommend the manuscript for publication after minor revision.

Auth 1.0 We thank the referee for their overall positive evaluation and specific feedback.

Ref 1.1 Eq 4 is given for the kinetic isotope effect (Line 78). It appears to be incorrect as one cannot obtain the reported values of ~1.010 to 1.050 given the reported epsilon values. The KIE (traditionally alpha) should be = (eps/1000) +1, or eps = (alpha – 1) * 1000.

Please be consistent with reporting eps and KIE values. This will likely require 1-2 sentences clarifying the definition of these parameters describing the isotope effect. Presumably, KIE values > 1 should have a positive eps value, whereas KIE < 1 have a negative eps value, after the equation above. In the current version of the manuscript, all reported KIE values in the current study are > 1, mostly negative eps values are reported in the Introduction (Lines 44, 111-114, etc.). This is likely due to reversal of the connotation, with heavy/light ratios of either the reaction substrates or products being in the numerator or denominator. In other words, whereas eps is reported from the perspective of the product of the reaction (negative value connotates that the product is depleted in the heavy isotope relative to substrate), the KIE values (i.e., alpha) are reported from the perspective of the substrate of the reaction, which becomes relatively enriched in the heavy isotope. For clarity, it would be useful to also report the eps values for the enzymes tested (in Table 2, if possible), consistent with literature cited in these Introduction lines.

Auth 1.1 Indeed, the definition of isotope effects can vary between different disciplines. We follow recommendations by Coplen (2011) as indicated in line 37 and define the kinetic isotope effect (KIE) in eq. 3, as the ratio of rate constants for the reaction of light vs. heavy isotopologues of O₂. This definition is in accordance with KIE values reported for O₂-consuming enzymes as referenced throughout the manuscript. The referee associated the term “isotope effect” with α (alpha), which is referred to as isotopic fractionation factor in Coplen (2011). We now include the inverse relationship between α and KIE, as defined above, in Eq. (4). As recommended by Coplen (2011) we refrain from adding the factor 1000, but otherwise we now display the same equation as given by the referee (α = ε+1). To improve clarity, we have included the fact that ε values are typically reported in permil in line 37. In summary, KIEs > 1, which is the case for all reported KIEs for O₂ consumption reactions, result in α values < 1 and negative ε values as reported in lines 44 and 114. As requested by the referee, we have now included ε values for the enzymes tested in Table 2 so that all our conversions are transparent and comparisons with different literature values are facilitated.

Line 37: “... isotopic fractionation … can be quantified, for example, with ¹⁸ε values (see Eq. (1)), which are typically reported in permil (‰) (Coplen, 2011):”

Lines 77-78: “Apparent ¹⁸O-KIEs are related to ¹⁸ε and ¹⁸α values as shown in Eq. (4). 

¹⁸O-KIE = (¹⁸α)^-1 = (¹⁸ε + 1)^-1”

Ref 1.2 Line 260 – How were the concentrations of organic substrate measured, as implied by this sentence? If only O2 concentrations were measured, please add text to clarify. It’s not clear if this is what is explained in Line 263-264. Presumably the initial substrate concentrations are assumed from experimental preparation and concentrations were not measured over time.

Auth 1.2 That is correct. We used the initial added organic substrate concentration to calculate K_m(S) as commonly done in enzyme kinetic studies. To clarify this, the manuscript text has been changed as indicated below.

Line 260: “… [i]_t is the initial (t=0), nominal concentration of an organic substrate (S) or the measured concentration of O₂ at time t, ...”

Ref 1.3. I suggest to improve the reaction mechanisms and Appendix equations, by better depicting the distribution of O₂ in the products of the reaction (see below). The appendix equations could be similarly color-coded as Fig. 6, for example.

Auth 1.3 The text in the appendix has been changed by colour coding O distribution in the products as suggested by the referee.

Ref 1.4 The discussion/conclusion justly describes how the findings of this study may be applied to delineate mechanisms of oxic, enzymatic respiration. It could be enhanced with discussion of other processes that presumably influence d18O of not only oxygen gas but also oxygen in oxidized, molecular end-products (e.g., D glucono- 1,5- lactone, Line 654; benzoquinone, Line 667; etc.). For example, I would appreciate to if the findings were discussed in the context of known isotope effects of biosynthesis (i.e., the reverse reaction of respiration).

Auth 1.4 As requested, we have extended our conclusion section by including a discussion on the O-isotopic composition of reaction products (see below). In the context of O₂-consuming enzymes, we consider two groups of products most relevant, namely O-containing aromatic compounds and H₂O₂. D-glucono-1,5-lactone, as suggested by the referee, does not incorporate oxygen atoms from O₂ during the glucose oxidase reaction. Its δ¹⁸O reflects the isotopic composition of the original glucose precursor and water molecules from earlier biosynthetic steps, rather than any fractionation associated with O₂ reduction. While hydroquinone, the precursor of benzoquinone, can be formed by oxygenase enzymes, we are not aware of any reported measurements of O-isotopic composition of hydroquinone or benzoquinone. We have thus not included this example specifically but rather discuss O-containing aromatic compounds in general.

Lines 569 ff: “… is not possible. In contrast to the differences observed for different active site structures, the ranges of ¹⁸O-KIEs associated with oxygenases (1.009-1.030) and oxidases (1.010-1.057) overlap. Nevertheless, these ranges provide benchmarks for comparisons with the O-isotopic composition of the main products of these enzymes, namely O-containing aromatic compounds and H₂O₂, respectively. δ¹⁸O values of natural, aromatic compounds, in which O-atoms primarily origin from O₂, have been measured to be 5-19 ‰ (Schmidt et al. 2001, https://doi.org/10.1016/S0031-9422(01)00017-6). Assuming a constant pool of dissolved O₂ with a δ¹⁸O value of 24 ‰ suggests underlying ¹⁸ε values for the biosynthesis of these compounds in the range of -5 to -19 ‰, which agrees well with the range of ¹⁸ε values (-9 to -30 ‰) reported in this and previous studies for oxygenase enzymes. For H₂O₂, measurements of O-isotopic composition in natural waters are scarce. In rainwater, δ¹⁸O values of H₂O₂ were 22-53 ‰ (Savarino and Thiemens 1999, https://doi.org/10.1016/S1352-2310(99)00122-3). Consequently, H₂O₂ is more enriched in ¹⁸O than expected from ¹⁸ε values of oxidase reactions (-9 to -53 ‰). However, this is not surprising considering that H₂O₂ can also be formed through different processes and rapidly reacts further, which will likely lead to an increase in δ¹⁸O values as observed. Overall, …”

Ref 1.5 Line 19 – change “which” to “associated with”

Auth 1.5 The text has been changed accordingly.

Line 19: “... displayed a narrower range of ¹⁸O-KIEs, with overall lower values (from 1.009 to 1.028), associated with an increase in the degree of ...”

Ref 1.6 Line 114 – change 18O-e to 18eps, or the variable 18O-eps needs to be defined.

Auth 1.6 We thank the referee for their attention to detail. The notation “¹⁸O-ε” should indeed be “¹⁸ɛ”. The variable “¹⁸ɛ” is now consistently used throughout the manuscript.

Line 114: “… values of -9 ‰ to ‐50 ‰, significantly exceeding the previously mentioned range of ¹⁸ε values observed for respiratory O₂…”

Ref 1.7 Fig. 3 – What is the “S” that is reduced/oxidized? Could the oxidized form of S be in red font?

Auth 1.7 To clarify, the final sentence of the figure caption has been changed as shown below. Because oxygen atoms from O₂ are not incorporated into the oxidized substrate (S_ox) during oxidase catalyzed reactions, the font color was not changed.

Figure 3. “… by oxidases. S_red and S-H represent an organic substrate before oxidation by an oxidase or monooxygenase, respectively, while S_ox and S-O(H) represent the corresponding organic reaction products.”

Ref 1.8 Fig. 6 – The red text appears to track oxygen atoms originating from O₂ in the reaction. Should the O in H₂O also be red?

Auth 1.8 We appreciate this suggestion and have colored the oxygen atom in H₂O in Fig. 6 red to consistently track oxygen atoms originating from O₂.

Comments by the referee are marked as “Ref {comment no.}”, answers by the authors are given as “Auth {comment no.}”. The text extracts following author comments show the revised parts of the manuscript with line numbers corresponding to the original submission.

Ref 2.0 This is a very interesting paper that impacts both fundamental enzymology/ biochemistry and environmental sciences.

The extension of oxygen kinetic isotope effects (KIEs) to a wide scope of enzymes, as well as the availability of comparative values for ¹⁸O and ¹⁷O isotope effects introduces a comprehensive resource for researchers. The authors are to be congratulated.

The experimental measurements are carefully collected and for the most part meaningfully interpreted.

The Discussion however could be improved after consideration of each of the comments below. Response/ revision to address these issues is important, prior to acceptance for publication.

Auth 2.0 We thank the referee for their overall positive evaluation and input for improving our discussion section.

Ref 2.1 pp. 16-17. The interpretation of kinetic oxygen isotope effects rest on the fact that these are competitive measurements and therefore always reflect kcat/Km parameters. Thus, the ¹⁸O KIE is reflective on all steps from O₂ binding up to and including the first irreversible step. If activation of O2 is multi-step, many steps can be reflected in the measurement. However, once an irreversible step has taken place during the ¹⁸O measurement, the value will be independent of subsequent, kinetically significant steps. For this reason, there can (and often are) different rate limiting steps when reporting on kcat/Km vs kcat.

Auth 2.1 We have specified the steps that are covered by measurements of ¹⁸O-KIEs more carefully in lines 74-75 and 400-403 (see below). Generally, we included comparisons with other studies that also report competitive ¹⁸O-KIEs and thus effects on kcat/Km for interpretations of reaction mechanisms relating to O₂ activation. The study we included in lines 404-405 was indeed an exception and upon closer examination, with the referee’s comment in mind, does not constitute a contradiction as stated in the original submission. We have thus removed this sentence to avoid confusion.

Lines 74-75: “Experimentally determined ¹⁸O-KIEs reflect the O-isotopic fractionation occurring in all elementary reaction steps beginning with interaction of enzyme with O₂ up to, and including, the first irreversible step (Roth 2007, https://doi.org/10.1016/j.cbpa.2007.01.683), which is often rate-limiting.”

Lines 400-405: “When comparing experimental ¹⁸O-KIEs to calculated ¹⁸O-EIEs, it is generally assumed that a measured ¹⁸O-KIE (i) reflects intrinsic ¹⁸O-KIEs of all electron and proton transfer steps up to, and including, the rate-limiting (i.e., first irreversible) step and (ii) is similar to, but not larger than, the ¹⁸O-EIE calculated for the formation of the product/intermediate after the rate-limiting step (Roth and Klinman 2005; Roth 2007). Based on these ¹⁸O-EIEs, the reduction of O₂ by KMO is thus likely characterized by a rate-limiting O₂^•- or FLOO^- formation.”

Ref 2.2 pp 16-17. The magnitude of a kinetic isotope effects has an additional component than the equilibrium isotope effects. This is because a KIE also contains a reaction coordinate frequency that can be altered (to some extent) by isotopic labelling {cf. Angeles-Boza, Chem Science 5, 1141 (2014)}.

Auth 2.2 We have specified this difference between KIEs and EIEs in lines 80-82, where this comparison first comes up.

Lines 80-82: “Because ¹⁸O-KIEs contain an additional reaction coordinate frequency compared to ¹⁸O-EIEs, intrinsic ¹⁸O-KIEs can be difficult to calculate (Roth 2007). Therefore, calculated ¹⁸O-EIEs are often used as a reference to assign experimentally determined ¹⁸O-KIEs to a specific rate-limiting step (Roth and Klinman 2005).”

Ref 2.3 p 18.  It is difficult to make a direct comparison between kcat and kcat/Km because you are comparing rate constants with different units, s^-1 and M^-1s^-1, respectively. The best way to think about the impact of the affinity of O2 on the ¹⁸O KIE is through the expression:

kobs = k1k2/ (k-1 +k2)

where k1 is the binding rate constant, k-1 is the off rate constant and k2 is the chemical step. If the off rate is slow (tightly bound O2?) then the ¹⁸O will only reflect k1. If k-1 is fast, binding approximates an equilibrium situation and the kobs is Kdk2.

It is very curious and interesting that the largest values in Table 4 occur for the reactions with the smaller Km. This may be the result of a small k-1 combined with a rate limiting binding step that is accompanied by electron transfer

Auth 2.3 In lines 430-455, we compared kcat and kcat/Km not quantitatively, but on a more conceptual basis, similar to the treatment in Northrop 1998. We understand, however, that this description can lead to misunderstandings and have revised this section based on the referee’s comment above. Our revised manuscript also contains an additional appendix (see PDF attached), providing mathematical considerations for this section that we consider to be relevant only to expert readers.

Lines 430 ff: “For KMO, cholesterol, choline, and glycolate oxidase, as well as glucose oxidase with 3 different substrates, which we consider to share a common reaction mechanism, we found a tentative correlation between ¹⁸O-KIEs and the corresponding K_m(O₂) values … Since ¹⁸O-KIEs reflect the ratios of reaction rates of the different O₂ isotopologues, a correlation between ¹⁸O-KIE and K_m(O₂) only makes sense when we consider the kinetic properties of the Michaelis constant (Northrop 1998). In O₂-consuming enzymes, O₂ typically binds to the enzyme after binding of the organic substrate (oxygenases), or in a ping-pong mechanism (oxidases) (Malmstrom 1982; Romero et al. 2018). Thus, we can describe the consumption of O₂ by these enzymes kinetically with a two-step reaction, where O₂ first binds reversibly to the enzyme, followed by an irreversible reduction step of O₂. In such a case, the measured ¹⁸O-KIE depends on the intrinsic ¹⁸O-KIE and ¹⁸O-EIE of the O₂ binding step, the ¹⁸O-KIE of the irreversible reduction step, and the forward commitment to catalysis. This commitment factor is the ratio of two elementary reaction rates, namely the rate of the irreversible reduction step divided by the rate of the backward reaction of O₂ binding (see Appendix D for details). In fact, as long as the reduction step is slower than the backward binding step, and thus the commitment factor below 1, the measured ¹⁸O-KIE will show an apparently linear trend with an increasing commitment factor, similar to the trend observed in Fig. 4. For this set of enzymes, it thus appears that K_m(O₂) is a proxy for the forward commitment to catalysis or the extent to which O₂ binding contributes to the overall reaction rate. One can indeed mathematically relate K_m(O₂) to the commitment factor, as shown in Appendix D, and reconcile the observed decrease in ¹⁸O-KIE with increasing K_m(O₂) values, if (i) O₂ binding and unbinding is faster than O₂ reduction for all enzymes but with different degrees of forward commitment, (ii) the intrinsic ¹⁸O-KIE for O₂ reduction is larger than for O₂ binding while all intrinsic isotope effects are close to identical for these enzymes, and (iii) the dissociation constant (the ratio of backward and forward reaction rates of O₂ binding) of these enzymes varies much less than K_m(O₂). If O₂ binding does not contribute to the overall rate, the apparent ¹⁸O-KIE is expected to reflect the intrinsic ¹⁸O-KIE of the rate-limiting O₂ reduction step. Accordingly, … lower ¹⁸O-KIEs (1.019-1.0.23), particularly for cholesterol, choline, and glycolate oxidase, can thus still arise from a rate-limiting O₂^•- or FLOO^- formation, but with increasing contributions from a relatively slower O₂ binding to the overall reaction rate that is likely associated with an intrinsic isotope effect close to unity because, upon binding, no bond changes occur in O₂.”

Ref 2.4 p.19. It is very interesting that there is a single example where the lambda value for comparison of ¹⁸O to ¹⁷O KIEs deviates from expectation. Since the two isotopes of oxygen have a different spin, this may suggest an unexpected spin component in the reaction.

Auth 2.4 This is indeed an interesting possibility. However, there is no evidence, as far as we know, for a possible reaction step associated with the suggested reaction mechanisms of flavin-dependent enzymes that would point towards such an unexpected spin component. As we already stated in line 477, and given the breath of our current study, we retain our opinion that “this reduction mechanism cannot be further elucidated in this study”. No changes were made.

Ref 2.5 In comparing the 18O values for Cu and Fe enzymes, the authors may wish to take a look at the different 18O EIEs for model Fe and Cu dependent systems (Tian and Klinman, JACS 114, 7117 (1993).

Auth 2.5 We have included a reference to Tian and Klinman (1993, https://doi.org/10.1021/ja00073a001) in lines 528-530, where we discuss KIEs for iron-dependent enzymes. For copper-dependent enzymes, the one additional experimental value provided in Tian and Klinman (1993) does not change the range of reported values, which we already gathered from more recent studies. Thus, we have not included this reference in the section discussing isotope effects of copper-dependent enzymes.

Lines 528-530: “Calculated or measured ¹⁸O-EIEs are also similar in magnitude, with 1.004-1.009 for iron-superoxo formation, 1.011-1.017 for iron-hydroperoxo formation, and 1.029 for iron-oxo formation (Tian and Klinman 1993; Mirica et al. 2008).”