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.

硝酸盐是整个地球生物圈中丰富的营养物质和电子受体。自然界中几乎所有的硝酸盐都是由亚硝酸盐氧化还原酶 (NXR) 多蛋白复合物氧化亚硝酸盐产生的。NXR 是全球生物氮循环中的关键酶，存在于产生环境中大部分硝酸盐的亚硝酸盐氧化细菌（包括 comammox 生物）和产生一半硝酸盐的厌氧氨氧化（anammox）细菌中。我们大气中的氮气。然而，尽管它在生物学中的核心作用和数十年的深入研究，但没有关于 NXR 的结构信息可用。在这里，我们对来自 anammox 细菌Kuenenia stuttgartiensis的 NXR 进行结构和生化分析，集成了 X 射线晶体学、冷冻电子断层扫描、螺旋重建冷冻电子显微镜、相互作用和重组研究以及酶动力学。我们发现 NXR 催化亚硝酸盐氧化和硝酸盐还原，并表明在细胞中，NXR 排列在数百纳米长的小管中。我们揭示了小管结构，并表明小管形成是由以前未知的含血红素亚基 NXR-T 诱导的。结果还揭示了酶活性位点的意外特征，蛋白质电子传递链中不寻常的辅因子配位，并阐明了复合物中的电子传递途径。