F. M . de Carvalho, Carolina. Isotopic fractionation of molecular oxygen during enzymatic O2-consuming processes in aquatic environments. 2024, Doctoral Thesis, University of Basel, Faculty of Science.
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Abstract
Stable isotope analysis of O2 has emerged as a valuable tool for studying O2 dynamics across various environmental scales, yet there is a lack of fundamental understanding regarding the large variability observed in isotopic fractionation of O2 during biological O2 consumption. Specifically, a wide range of 18O enrichment factors (18ε) from -29 to -1 ‰ has been reported for respiration in aquatic environments, while 18O-kinetic isotope effects (18O-KIEs) ranging from 1.009 to 1.053 (equivalent to 18ε values of -9 ‰ to -50 ‰) have been observed for 26 O2-consuming enzymes. As all biological O2 consumption is performed by O2-consuming enzymes, the variability in isotopic fractionation of O2 at the enzyme level likely contributes to the observed variability in 18ε values. Understanding O2 isotopic fractionation at the enzyme level can provide insights into the underlying sources of 18ε value variability for biological O2 consumption, enhancing the accuracy and reliability of stable isotope analysis of O2 for studying O2-consumption dynamics in aquatic environments. However, stable isotope analysis of O2 is hindered by the lack of reference materials for isotope ratio calibration, often relying on a one-point calibration with ambient air, which can lead to substantial measurement uncertainties. Hence, this thesis aimed to first develop methodologies for improving the measurement precision and accuracy of stable isotope analysis of O2 (Chapter 2), second, investigate the mechanisms driving the variability in isotopic fractionation of O2 at the enzyme level (Chapter 3), and lastly, to provide insights on the effect on O2 isotopic fractionation originally caused by O2 consuming enzymes at the environment level (Chapter 4).
In Chapter 2, a multi-point isotope-ratio calibration approach was developed using spinach leaf thylakoids and source waters with different δ18O and δ17O values to photosynthetically produce O2 with known isotopic compositions. By comparing the δ18O and δ17O values of the source water and produced O2, measurement errors and δ scale correction factors were determined. While no significant bias was observed on the δ18O scale (in a range of δ18O values from -56 to +95 ‰, maximum error 0.8 ‰), a δ17O scale compression was evident (in a range of δ17O values from -30 to +46 ‰, maximum error 3.3 ‰), indicating the need for δ17O scale correction.
In Chapter 3, nineteen 18O-KIEs were determined for several flavin- and copper-dependent oxidases, as well as for one flavin-dependent monooxygenase and one heme-copper dependent oxidase to gain insights into the mechanistic origins of the observed variability in 18O-KIEs. Distinct patterns of isotopic fractionation of O2 were revealed within and between enzyme groups, reflecting differences in active site structures and O2 reduction mechanisms. Both flavin- and copper-dependent enzymes exhibited two distinct ranges of 18O-KIEs associated with two different O2 reduction mechanisms. However, iron- and copper-dependent enzymes displayed a narrower range of lower 18O-KIEs than flavin-dependent enzymes, increasing with the degree of O2 reduction. These findings support generalizations of expected 18O-KIEs for other enzymes and potentially aid in interpreting stable isotopic fractionation across environmental scales.
Finally, Chapter 4 investigated whether microbial ammonia and methane oxidation could contribute to the observed discrepancy in biological O2 consumption 18ε values between laboratory incubations (-18 to -22 ‰) and in situ measurements in aquatic environments (-10 to -18 ‰). By performing experiments with three methanotrophic bacteria and a comammox bacterium we could estimate the in vivo 18ε values of soluble methane monooxygenase (sMMO), particulate methane monooxygenase (pMMO), and ammonia monooxygenase (AMO). The estimated 18ε values for sMMO, pMMO and AMO (-19 ± 1 ‰ to -24 ± 4 ‰) were not significantly different from typical heterotrophic respiration 18ε values. These results indicate that although substantial, O2 consumption by sMMO, pMMO, or AMO cannot explain the observed discrepancy in 18ε values between laboratory incubations and in situ measurements, but provide insights into their potential reaction mechanisms.
In summary, this work demonstrates that a comprehensive understanding of isotopic fractionation of O2 at the enzyme level, coupled with improved calibration methodologies, can significantly enhance our fundamental and mechanistic understanding of the variability in O2 isotopic fractionation during biological O2 consumption. This, in turn, can improve the reliability and accuracy of stable isotope analysis as a tool for studying important O2-consuming processes, providing deeper insights into O2 consumption dynamics across different environmental contexts and contributing to more accurate modeling of biological O2 consumption and ecosystem health assessments.
In Chapter 2, a multi-point isotope-ratio calibration approach was developed using spinach leaf thylakoids and source waters with different δ18O and δ17O values to photosynthetically produce O2 with known isotopic compositions. By comparing the δ18O and δ17O values of the source water and produced O2, measurement errors and δ scale correction factors were determined. While no significant bias was observed on the δ18O scale (in a range of δ18O values from -56 to +95 ‰, maximum error 0.8 ‰), a δ17O scale compression was evident (in a range of δ17O values from -30 to +46 ‰, maximum error 3.3 ‰), indicating the need for δ17O scale correction.
In Chapter 3, nineteen 18O-KIEs were determined for several flavin- and copper-dependent oxidases, as well as for one flavin-dependent monooxygenase and one heme-copper dependent oxidase to gain insights into the mechanistic origins of the observed variability in 18O-KIEs. Distinct patterns of isotopic fractionation of O2 were revealed within and between enzyme groups, reflecting differences in active site structures and O2 reduction mechanisms. Both flavin- and copper-dependent enzymes exhibited two distinct ranges of 18O-KIEs associated with two different O2 reduction mechanisms. However, iron- and copper-dependent enzymes displayed a narrower range of lower 18O-KIEs than flavin-dependent enzymes, increasing with the degree of O2 reduction. These findings support generalizations of expected 18O-KIEs for other enzymes and potentially aid in interpreting stable isotopic fractionation across environmental scales.
Finally, Chapter 4 investigated whether microbial ammonia and methane oxidation could contribute to the observed discrepancy in biological O2 consumption 18ε values between laboratory incubations (-18 to -22 ‰) and in situ measurements in aquatic environments (-10 to -18 ‰). By performing experiments with three methanotrophic bacteria and a comammox bacterium we could estimate the in vivo 18ε values of soluble methane monooxygenase (sMMO), particulate methane monooxygenase (pMMO), and ammonia monooxygenase (AMO). The estimated 18ε values for sMMO, pMMO and AMO (-19 ± 1 ‰ to -24 ± 4 ‰) were not significantly different from typical heterotrophic respiration 18ε values. These results indicate that although substantial, O2 consumption by sMMO, pMMO, or AMO cannot explain the observed discrepancy in 18ε values between laboratory incubations and in situ measurements, but provide insights into their potential reaction mechanisms.
In summary, this work demonstrates that a comprehensive understanding of isotopic fractionation of O2 at the enzyme level, coupled with improved calibration methodologies, can significantly enhance our fundamental and mechanistic understanding of the variability in O2 isotopic fractionation during biological O2 consumption. This, in turn, can improve the reliability and accuracy of stable isotope analysis as a tool for studying important O2-consuming processes, providing deeper insights into O2 consumption dynamics across different environmental contexts and contributing to more accurate modeling of biological O2 consumption and ecosystem health assessments.
Advisors: | Lehmann, Moritz F and Pati, Sarah |
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Committee Members: | Seebeck, Florian Peter and Elsner, Martin |
Faculties and Departments: | 05 Faculty of Science > Departement Chemie > Chemie > Molecular Bionics (Seebeck) 05 Faculty of Science > Departement Umweltwissenschaften > Geowissenschaften > Aquatic and Isotope Biogeochemistry (Lehmann) |
UniBasel Contributors: | Pati, Sarah and Seebeck, Florian Peter |
Item Type: | Thesis |
Thesis Subtype: | Doctoral Thesis |
Thesis no: | 15537 |
Thesis status: | Complete |
Number of Pages: | 144 |
Language: | English |
Identification Number: |
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edoc DOI: | |
Last Modified: | 16 Nov 2024 05:30 |
Deposited On: | 15 Nov 2024 12:26 |
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