# Hydrazine Synthase From Anammox Is Inhibited by Linear and Aromatic Alkynes

## TL;DR

Linear alkynes inhibit anammox bacteria by targeting hydrazine synthase, a key enzyme in their metabolism.

## Contribution

This study identifies hydrazine synthase as the target of alkynes in anammox bacteria, enabling selective inhibition for measuring N2O contributions.

## Key findings

- C2–C5 alkynes inhibit anammox activity by targeting hydrazine synthase.
- Larger or aromatic alkynes do not inhibit anammox activity.
- 1-octyne is a suitable selective inhibitor for distinguishing N2O sources in mixed environments.

## Abstract

N2O emissions by nitrifiers are often estimated using selective inhibitors, such as 1‐alkynes. However, the effects of these inhibitors on anaerobic ammonium‐oxidising (anammox) bacteria are largely unknown. In this study, we assessed the inhibitory effect of linear and aromatic alkynes on anammox activity and identified their target enzyme. ‘Candidatus Kuenenia stuttgartiensis’ biomass and constructed wetland soil samples were incubated with 10 μM of C2–C₈ linear alkynes or phenylacetylene for 10 days. Anammox activity was determined using the isotopic tracer 15N‐nitrite and 29N2 production. Anammox activity was suppressed by C2–C5 alkynes, whereas the larger or aromatic alkynes caused no inhibition. However, hydrazine oxidation activity was not affected, indicating that C2–C5 alkynes inactivated enzymes upstream of hydrazine dehydrogenase. In incubations using an NO donor and 15N‐ammonium, 29N2 production stopped, suggesting that hydrazine synthase was the target of these alkynes. A comparable trend was observed in the wetland samples, but with a less pronounced reduction in 29N2 production. Since alkynes >C5 did not affect anammox, these findings demonstrate the suitability of using 1‐octyne as a selective inhibitor to quantify N2O contributions from ammonia‐oxidising bacteria (AOB) versus ammonia‐oxidising archaea (AOA) in oxic/anoxic interface environments.

Linear and aromatic alkynes inhibit anammox in culture and in wetlands. The inhibition by alkynes is specific to the hydrazine synthase, a key enzyme in the anammox pathway.