Nitric oxide-forming nitrite reductases in the anaerobic ammonium oxidizer Kuenenia stuttgartiensis

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria contribute to the global nitrogen cycle by removing fixed nitrogen from the environment. They do so via the anaerobic oxidation of ammonium to dinitrogen gas, with nitrite as terminal electron acceptor. The first step in this so-called anammox reaction is the conversion of nitrite to nitric oxide by nitrite reductase. Next, nitric oxide is combined with ammonium to form hydrazine by hydrazine synthase, after which hydrazine is oxidized to dinitrogen gas by hydrazine dehydrogenase. In contrast to the other catabolic anammox enzymes, different anammox species encode different potential nitrite reductase enzymes. On top of that, there is a redundancy in genes encoding for nitrite reductase in single anammox species. The unusual diversity and redundancy in anammox nitrite reductases is unexplained. The genome of the model anammox species "Candidatus Kuenenia stuttgartiensis" encodes for three putative nitrite reductases. Here, we investigated which of these nitrite reductases is or are active in K. stuttgartiensis. Active nitric oxide-producing nitrite reductases were enriched from K. stuttgartiensis cells via fast protein liquid chromatography. Nitric oxide production by the enriched nitrite reductases was followed with membrane inlet mass spectrometry. Combining the activity assays with proteomics analysis indicated that the soluble nitrite reductases NirS and HAOr most strongly correlated with enzyme activity. This indicates that K. stuttgartiensis employs two distinct nitrite reductases to keep its nitric oxide pool replenished.