Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

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.

All values of d17O and d18O were converted to a VSMOW2-SLAP2 scale using a two point calibration of O2 liberated from VSMOW2 and SLAP2 standards using a CoF3 reactor as previously described (Schoenemann et al., 2013; Barkan and Luz, 2005). All slopes (i.e., k values) and slope uncertainties were calculated as linear fits to plots of d17O vs. d18O using the polyfit function in MATLAB (linear least-squares). Typical reproducibility of Ar-free lab air is 0.005‰ for D'17O and 0.02‰ 0.02‰ for d18O. The oxygen isotope results presented in this study are the mean and standard deviation when the number of samples receiving the exact same treatment is greater than one. In situations where replicates were not possible (e.g., measurements along a reaction progress array), uncertainty is assumed to be equal to the reproducibility of lab air reported above.

- Imported original file "Sutherland GCA table.xlsx" into the BCO-DMO system.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved the final file as "923859_v1_reactive_o2_species.csv".

NSF Award Abstract:
The balance between photosynthetic production of oxygen and biological consumption of oxygen in the marine environment plays a critical role in regulating the composition of Earth’s atmosphere and the long-term stability of Earth’s climate. The ability to accurately measure the production and consumption of oxygen in the marine environment is central to building an informed understanding of the past, present, and future of oxygen and Earth’s climate. Measurement of the abundance of different oxygen isotopes (i.e. oxygen-16, oxygen-17, and oxygen-18) in dissolved oxygen in seawater is a powerful analytical tool that can be used to determine the magnitude of photosynthesis and biological oxygen consumption. This is possible because biological reactions that produce or consume oxygen tend to preferentially utilize different isotopes of oxygen. This analytical tool has been used for two decades to investigate ocean primary productivity, however, there is still significant uncertainty in how biogeochemical processes such as respiration preferentially select and utilize different oxygen isotopes. To remedy this uncertainty, researchers at Harvard University will perform a lab-based calibration and sea-going field deployment of emerging oxygen isotope analyses that target molecules of oxygen that contain two rare oxygen isotopes inside of one molecule (a.k.a. “clumps”, i.e. 17O18O and 18O18O). This work will quantify the clumped oxygen isotope signatures of several of the most consequential oxygen-involving reactions that occur in the marine ecosystem. These oxygen isotope signatures will be used to refine current methods and assumptions for the measurement of gross oxygen production in the global ocean. This research will also help train the next generation of Earth scientists through the mentorship of one graduate student and two undergraduate students. This project will also facilitate the participation of researchers in content creation for a national science competition for middle and high school students that reaches thousands of students annually.

The application of clumped O2 isotope measurements to dissolved oxygen in seawater is poised to give a greater mechanistic view of gross primary productivity and marine oxygen cycling than previously attainable. To realize the analytical potential of clumped oxygen isotope methods, these researchers will characterize the clumped oxygen isotope effects associated with enzyme-level reactions, whole organisms, and the marine water column. Enzyme-level studies will include isotope characterization of a terminal-O2 reductase and enzymes that metabolize reactive oxygen species such as superoxide and hydrogen peroxide. Organism studies will target common and numerically abundant phototrophs and heterotrophs to explore the expected breadth of isotope signatures and fractionation factors in the marine water column. Marine water column dissolved oxygen samples will be collected during one of multiple candidate cruises and analyzed using both traditional triple oxygen isotope techniques and newly developed clumped oxygen isotope methods. Lastly, they will employ the results of lab-based study of clumped O2 to build a model for the interpretation of environmental data with the aim of improving the accuracy and precision of field measurements of gross primary production.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.