Hydrazines as Substrates and Inhibitors of the Archaeal Ammonia Oxidation Pathway

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) perform key steps in the global nitrogen cycle, the oxidation of ammonia to nitrite. While the ammonia oxidation pathway is well characterized in AOB, many knowledge gaps remain about the metabolism of AOA. Hydroxylamine is an intermediate in both AOB and AOA, but homologues of hydroxylamine dehydrogenase (HAO), catalyzing bacterial hydroxylamine oxidation, are absent in AOA. Hydrazine is a substrate for bacterial HAO, while phenylhydrazine is a suicide inhibitor of HAO. Here, we examine the effect of hydrazines in AOA to gain insights into the archaeal ammonia oxidation pathway. We show that hydrazine is both a substrate and an inhibitor for AOA and that phenylhydrazine irreversibly inhibits archaeal hydroxylamine oxidation. Both hydrazine and phenylhydrazine interfered with ammonia and hydroxylamine oxidation in AOA. Furthermore, the AOA “Candidatus Nitrosocosmicus franklandus” C13 oxidized hydrazine into dinitrogen (N(2)), coupling this reaction to ATP production and O(2) uptake. This study expands the known substrates of AOA and suggests that despite differences in enzymology, the ammonia oxidation pathways of AOB and AOA are functionally surprisingly similar. These results demonstrate that hydrazines are valuable tools for studying the archaeal ammonia oxidation pathway. IMPORTANCE Ammonia-oxidizing archaea (AOA) are among the most numerous living organisms on Earth, and they play a pivotal role in the global biogeochemical nitrogen cycle. Despite this, little is known about the physiology and metabolism of AOA. We demonstrate in this study that hydrazines are inhibitors of AOA. Furthermore, we demonstrate that the model soil AOA “Ca. Nitrosocosmicus franklandus” C13 oxidizes hydrazine to dinitrogen gas, and this reaction yields ATP. This provides an important advance in our understanding of the metabolism of AOA and expands the short list of energy-yielding compounds that AOA can use. This study also provides evidence that hydrazines can be useful tools for studying the metabolism of AOA, as they have been for the bacterial ammonia oxidizers.