Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N(2)) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N(2)H(4)). Here we show that N(2)H(4) is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N(2)H(4). We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N(2)H(4) synthesis and its oxidation to N(2). These results present a new biochemical reaction forging an N-N bond and fill a lacuna in our understanding of the biochemical synthesis of the N(2) in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

Anaerobic ammonium-oxidizing (anammox) bacteria primarily grow by the oxidation of ammonium coupled to nitrite reduction, using CO2 as the sole carbon source. Although they were neglected for a long time, anammox bacteria are encountered in an enormous species (micro)diversity in virtually any anoxic environment that contains fixed nitrogen. It has even been estimated that about 50% of all nitrogen gas released into the atmosphere is made by these 'impossible' bacteria. Anammox catabolism most likely resides in a special cell organelle, the anammoxosome, which is surrounded by highly unusual ladder-like (ladderane) lipids. Ammonium oxidation and nitrite reduction proceed in a cyclic electron flow through two intermediates, hydrazine and nitric oxide, resulting in the generation of proton-motive force for ATP synthesis. Reduction reactions associated with CO2 fixation drain electrons from this cycle, and they are replenished by the oxidation of nitrite to nitrate. Besides ammonium or nitrite, anammox bacteria use a broad range of organic and inorganic compounds as electron donors. An analysis of the metabolic opportunities even suggests alternative chemolithotrophic lifestyles that are independent of these compounds. We note that current concepts are still largely hypothetical and put forward the most intriguing questions that need experimental answers.

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Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species.

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (−750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.