Isotopic Fractionation by a Fungal P450 Nitric Oxide Reductase during the Production of N₂O

Abstract

Nitrous oxide (N2O) is a potent greenhouse gas with a 100-year global warming potential approximately 300 times that of CO2. Because microbes account for over 75% of the N2O released in the U.S., understanding the biochemical processes by which N2O is produced is critical to our efforts to mitigate climate change. In the current study, we used gas chromatography-isotope ratio mass spectrometry (GC-IRMS) to measure the δ(15)N, δ(18)O, δ(15)N(α), and δ(15)N(β) of N2O generated by purified fungal nitric oxide reductase (P450nor) from Histoplasma capsulatum. The isotope values were used to calculate site preference (SP) values (difference in δ(15)N between the central (α) and terminal (β) N atoms in N2O), enrichment factors (ε), and kinetic isotope effects (KIEs). Both oxygen and N(α) displayed normal isotope effects during enzymatic NO reduction with ε values of -25.7‰ (KIE = 1.0264) and -12.6‰ (KIE = 1.0127), respectively. However, bulk nitrogen (average δ(15)N of N(α) and N(β)) and N(β) exhibited inverse isotope effects with ε values of 14.0‰ (KIE = 0.9862) and 36.1‰ (KIE = 0.9651), respectively. The observed inverse isotope effect in δ(15)N(β) is consistent with reversible binding of the first NO in the P450nor reaction mechanism. In contrast to the constant SP observed during NO reduction in microbial cultures, the site preference measured for purified H. capsulatum P450nor was not constant, increasing from ∼ 15‰ to ∼ 29‰ during the course of the reaction. This indicates that SP for microbial cultures can vary depending on the growth conditions, which may complicate source tracing during microbial denitrification.

Isotopic evidence for nitrous oxide production pathways in a partial nitritation-anammox reactor

“…There is a strong correlation between d 18 O and SP (R 2 ¼ 0.53), and the difference between d 18 O and SP (0.2e16‰) is lowest when NO À 2 is added before anoxia is introduced. This is also in agreement with the results of the previous subsection and again suggests a shift between two dominant pathways with little influence from dependence of fractionation factors on varying conditions or O-isotope exchange (Buchwald and Yang et al, 2014). When the air flow was switched to N 2 , however, SP increased immediately by~9‰ as N 2 O production by AOB ceased (Yu et al, 2010).…”

“…3 The d 18 O and SP values show a strong correlation (R 2 ¼ 0.72), which suggests minimal influence from O-isotope exchange, which would be expected to alter d 18 O with no effect on SP. The difference between d 18 O and SP varies between À7.8 and 20‰ with the smallest differences during NO À 2 addition, which suggests a shift between two dominant N 2 O production pathways, with little influence from confounding factors such as the dependence of fractionation factors on process conditions (Buchwald and Yang et al, 2014). During cycle o, the NH þ 4 setpoint was raised to 100 mg-N L À1 to simultaneously enhance N 2 O production via nitrifier denitrification and NH 2 OH oxidation (Wunderlin et al, 2013a;Ni et al, 2014).…”

Valence tautomerism in synthetic models of cytochrome P450

“…In this case, recent work shows that heme-thiolate model systems can catalytically hydroxylate inert C-H bonds using molecular O 2 with >200 turnovers, i.e., bioinspired functional mimics of cytP450 (11). In addition, cytP450 enzymes form stable diamagnetic Fe III -NO adducts (12). In contrast, attempts to form stable Fe III -NO adducts of thiolate-bound iron porphyrin model systems inevitably results in the NO attacking the thiolate sulfur and not the iron.…”

Estimation of isotopologue variation of N<sub>2</sub>O during denitrification by <i>Pseudomonas aureofaciens</i> and <i>Pseudomonas</i> chlororaphis: Implications for N<sub>2</sub>O source apportionment

“…For details regarding this method see Yang et al (2014). These data were used to determine when the N2O concentration was sufficient for isotope analysis and to estimate the volume of headspace required for isotope analysis over the course of the reaction.…”

“…However, SP is not without variation (Toyoda et al, 2017). In addition to production pathway, numerous factors could theoretically control the degree of variation in SP including differences in bacterial species, the specific enzyme involved in its production (Yang et al, 2014), and, for Biogeosciences Discuss., https://doi.org /10.5194/bg-2017- produced by two denitrifier species in vitro, as well the effect of these factors on SP values of N2O. We conducted our study with Pseudomonas chlororaphis subsp.…”