Open Access

Tadeo Moreno Chicano, Lea Theresa Dietrich, Naomi M. de Almeida, Mohd Akram, Elisabeth Hartmann, Franziska Leidreiter, Daniel Leopoldus, Melanie Mueller, Ricardo Sánchez, Guylaine H. L. Nuijten, Joachim Reimann, Kerstin-Anikó Seifert, Ilme Schlichting, Laura van Niftrik, Michael S. M. Jetten, Andreas Dietl, Boran Kartal, Kristian Kurt Parey, Thomas R. M. Barends

• Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.