the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 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.
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RC1: 'Comment on egusphere-2025-1193', Anonymous Referee #1, 24 Apr 2025
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.
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.
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.
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.
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).Minor revisions:
Line 19 – change “which” to “associated with”
Line 114 – change 18O-e to 18eps, or the variable 18O-eps needs to be defined.
Fig. 3 – What is the “S” that is reduced/oxidized? Could the oxidized form of S be in red font?
Fig. 6 – The red text appears to track oxygen atoms originating from O2 in the reaction. Should the O in H2O also be red?
Citation: https://doi.org/10.5194/egusphere-2025-1193-RC1 -
AC1: 'Reply on RC1', Sarah G. Pati, 16 Jun 2025
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 Km(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 (Sox) during oxidase catalyzed reactions, the font color was not changed.
Figure 3. “… by oxidases. Sred and S-H represent an organic substrate before oxidation by an oxidase or monooxygenase, respectively, while Sox 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.
Citation: https://doi.org/10.5194/egusphere-2025-1193-AC1
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AC1: 'Reply on RC1', Sarah G. Pati, 16 Jun 2025
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RC2: 'Comment on egusphere-2025-1193', Anonymous Referee #2, 08 May 2025
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.
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.
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)}.
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
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.
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).
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AC2: 'Reply on RC2', Sarah G. Pati, 16 Jun 2025
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 Km(O2) values … Since 18O-KIEs reflect the ratios of reaction rates of the different O2 isotopologues, a correlation between 18O-KIE and Km(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 Km(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 Km(O2) to the commitment factor, as shown in Appendix D, and reconcile the observed decrease in 18O-KIE with increasing Km(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 Km(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).”
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AC2: 'Reply on RC2', Sarah G. Pati, 16 Jun 2025
Interactive discussion
Status: closed
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RC1: 'Comment on egusphere-2025-1193', Anonymous Referee #1, 24 Apr 2025
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.
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.
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.
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.
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).Minor revisions:
Line 19 – change “which” to “associated with”
Line 114 – change 18O-e to 18eps, or the variable 18O-eps needs to be defined.
Fig. 3 – What is the “S” that is reduced/oxidized? Could the oxidized form of S be in red font?
Fig. 6 – The red text appears to track oxygen atoms originating from O2 in the reaction. Should the O in H2O also be red?
Citation: https://doi.org/10.5194/egusphere-2025-1193-RC1 -
AC1: 'Reply on RC1', Sarah G. Pati, 16 Jun 2025
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 Km(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 (Sox) during oxidase catalyzed reactions, the font color was not changed.
Figure 3. “… by oxidases. Sred and S-H represent an organic substrate before oxidation by an oxidase or monooxygenase, respectively, while Sox 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.
Citation: https://doi.org/10.5194/egusphere-2025-1193-AC1
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AC1: 'Reply on RC1', Sarah G. Pati, 16 Jun 2025
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RC2: 'Comment on egusphere-2025-1193', Anonymous Referee #2, 08 May 2025
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.
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.
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)}.
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
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.
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).
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AC2: 'Reply on RC2', Sarah G. Pati, 16 Jun 2025
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 Km(O2) values … Since 18O-KIEs reflect the ratios of reaction rates of the different O2 isotopologues, a correlation between 18O-KIE and Km(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 Km(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 Km(O2) to the commitment factor, as shown in Appendix D, and reconcile the observed decrease in 18O-KIE with increasing Km(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 Km(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).”
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AC2: 'Reply on RC2', Sarah G. Pati, 16 Jun 2025
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