Dataset: The influence of reactive oxygen species on "‘respiration" isotope effect

This dataset has not been validatedData not availableVersion 1 (2024-03-28)Dataset Type:experimental

Principal Investigator: David Johnston (Harvard University)

BCO-DMO Data Manager: Shannon Rauch (Woods Hole Oceanographic Institution)


Project: Clumped Oxygen Isotope Signature of Marine Dissolved Oxygen (Microbial isotope effects)


Abstract

The triple-oxygen isotope (17O/16O, 18O/16O) measurement of oxygen-bearing species represents one of the most robust tools to directly trace oxygen cycling in the environment. One particularly consequential application of this isotope system is the analysis of dissolved oxygen (O2) in aquatic environments to determine gross oxygen production. This approach assumes that photosynthesis, microbial respiration, and gas exchange are the main drivers of dissolved O2 isotope compositions, and that each...

Show more

We have chosen a representative from each enzyme group to offer initial isotopic constraints on O2 evolved from superoxide dismutase (SOD) and catalase. Specifically, we analyzed a representative CuZn SOD (Sigma S5395) and typical catalase (heme-binding, clade 3, small subunit, Sigma C1345). The same experiment apparatus was used to investigate the isotope effects of both enzymes. The reaction vessel consisted of a closed-system glass bulb with an inlet subject to continuous He flow (pre-scrubbed with 5A molecular sieve at liquid nitrogen temperature) through a glass frit, an outlet stream, and an injection port outfitted with a blue butyl rubber septum. The outlet stream was passed through multiple loops of stainless steel tubing held at liquid nitrogen temperature to trap any water vapor that escaped the reactor. KO2 or H2O2 were added for reactions with SOD and catalase, respectively. Effluent He carrier gas and product O2 were then passed through a 5A molecular sieve trap to isolate O2 gas evolved by the enzyme of interest.

In the case of catalase, 2 milligrams (mg) of enzyme was dissolved in approximately 50 milliliters (mL) of ultra-high purity (18 MX MilliQ) water and bubbled with He for a minimum of one hour to remove O2, at which point 30% H2O2 was introduced to the solution through the injection port to an initial concentration of 3.9 millimolar (mM) in the reaction vessel. The reaction was allowed to proceed for a range of time intervals (a few minutes to several hours) to ensure adequate coverage of reaction progress for use with the Rayleigh equation. The oxygen isotope composition of the starting H2O2 was determined using the same experimental setup (without catalase) by adding a slurry containing MilliQ water and KMnO4 (amounting to a 10X excess of H2O2), which quantitatively oxidizes H2O2 to O2. The average O2 yield of the catalase and H2O2 treatment was 48% (n = 3) that of the KMnO4 treatment, consistent with the expected 2:1 stoichiometry of catalase.

SOD experiments were conducted with the same reaction vessel with some modifications. Given the rapid rate of reaction between superoxide and SOD, we instead characterized the oxygen isotope composition of the starting material (introduced as KO2) and the fully completed reaction to determine how oxygen isotopes are partitions between the oxidized and the reduced product. Methods for both measurements were modified from one previous study investigating the 18O isotope effects of CuZn SOD. To measure the initial ROS oxygen isotope composition, several milligrams of KO2 were introduced into the empty and dry reaction chamber, which was immediately flushed with He. Since water/moisture will cause KO2 to spontaneously disproportionate, the powder was kept dry prior to starting the reaction. Separately, a He-sparged solution containing MilliQ water and an excess of K3[Fe(CN)6] was prepared and added to the chamber to quantitatively oxidize the starting material to O2. The SOD disproportionation reaction was performed similarly but using a solution of CuZn SOD, horse-radish peroxidase (HRP, to reduce product H2O2 quantitatively to water), and K4[Fe(CN)6]∙3H2O (reducing equivalents for HRP; Smirnov and Roth, 2006). To further survey ROS reactions in the environment and their impact on the oxygen isotope systematics of dissolved O2, we examined one non-enzymatic ROS decay pathway - Fe-mediated H2O2 degradation. We used the methods outlined in Dole et al. (1952) with the same experimental apparatus described above to explore this Fe-catalyzed pathway. Reaction progress was monitored by comparing the amount of O2 evolved from the reaction vessel relative to that from the KMnO4 treatment.

Instruments:
Following O2 gas collection, the sample volume was sealed with gas-tight valves under He flow and immediately transferred to a gas purification manifold to be analyzed on a Thermo Scientific MAT 253 Plus isotope ratio mass spectrometer (IRMS) at Harvard University (Cowie and Johnston, 2016). Carrier He gas was pumped away from the still-frozen sample before thawing. Oxygen gas should be the only product gas evolved from these reactions; however, to ensure trace atmospheric contaminants were not present, each sample was passed through a 3 m gas chromatography (GC) column packed with 5A molecular sieve maintained at 30 °C with He carrier gas at 15 mL/min. Oxygen was collected from GC effluent on a U-trap containing 5A molecular sieve held at liquid nitrogen temperature. Since all sample O2 was generated from KO2 and H2O2, Ar - which can interfere with TOI analyses - was not present (Yeung et al., 2018; Ash et al., 2020). Finally, effluent O2 gas was cryo-focused and allowed to thaw at room temperature for a minimum of 20 min to ensure no isotope fractionation before introduction into the IRMS for analysis. Each analysis comprises the average value of four acquisition blocks, each consisting of 20 cycles between the reference gas and sample gas (total counting time on sample gas was 400 s per acquisition). Measurements were typically run at 5000 m V signal on the m/z 32 Faraday cup (3 x 10^8 resistor). Total acquisition time for a single analysis is roughly two hours.


Related Datasets

No Related Datasets

Related Publications

Results

Sutherland, K. M., Hemingway, J. D., & Johnston, D. T. (2022). The influence of reactive oxygen species on “respiration” isotope effects. Geochimica et Cosmochimica Acta, 324, 86–101. https://doi.org/10.1016/j.gca.2022.02.033
Methods

Ash, J. L., Hu, H., & Yeung, L. Y. (2019). What Fractionates Oxygen Isotopes during Respiration? Insights from Multiple Isotopologue Measurements and Theory. ACS Earth and Space Chemistry, 4(1), 50–66. https://doi.org/10.1021/acsearthspacechem.9b00230
Methods

Barkan, E., & Luz, B. (2005). High precision measurements of 17O/16O and 18O/16O ratios in H2O. Rapid Communications in Mass Spectrometry, 19(24), 3737–3742. Portico. https://doi.org/10.1002/rcm.2250
Methods

Cowie, B. R., & Johnston, D. T. (2016). High-precision measurement and standard calibration of triple oxygen isotopic compositions (δ18O, Δ17O) of sulfate by F2 laser fluorination. Chemical Geology, 440, 50–59. https://doi.org/10.1016/j.chemgeo.2016.07.003
Methods

Dole, M., Rudd, D. P., Muchow, G. R., & Comte, C. (1952). Isotopic Composition of Oxygen in the Catalytic Decomposition of Hydrogen Peroxide. The Journal of Chemical Physics, 20(6), 961–968. https://doi.org/10.1063/1.1700657