Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased significantly over the last decades 1-3 . As the final step of bacterial denitrification, the critical greenhouse gas N2O is reduced to chemically inert N2 (ref. 1,4) in a reaction catalyzed by the copper-dependent nitrous oxide reductase (N2OR) 5 . The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-bindingcassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL 4,6 .Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY, and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein features a binding site for a Cu + ion and interacts specifically with NosL in its nucleotide-free state, while its binding to N2OR requires a conformational change triggered by ATP binding.Mutually exclusive complex structures of NosDFY with NosL and N2OR reveal a sequential metal trafficking and assembly pathway for the most complex copper site known to date. In it, NosDFY acts as a mechanical energy transducer rather than a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.

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The mitochondrial Lon protease homolog (LonP1) hexamer controls mitochondrial health by digesting proteins from the mitochondrial matrix that are damaged or must be removed. Understanding how it is regulated requires characterizing its mechanism. Here, we show how human LonP1 functions, based on eight different conformational states that we determined by cryo-EM with a resolution locally extending to 3.6 Å for the best ordered states. LonP1 has a poorly ordered N-terminal part with apparent threefold symmetry, which apparently binds substrate protein and feeds it into its AAA+ unfoldase core. This translocates the extended substrate protein into a proteolytic cavity, in which we report an additional, previously unidentified Thr-type proteolytic center. Threefold rocking movements of the flexible N-terminal assembly likely assist thermal unfolding of the substrate protein. Our data suggest LonP1 may function as a sixfold cyclical Brownian ratchet controlled by ATP hydrolysis.

Most proteins found in the outer membrane of Gram-negative bacteria share a common domain: the transmembrane beta-barrel. These outer membrane -barrels (OMBBs) occur in multiple sizes, and different families with a wide range of functions evolved independently by amplification from a pool of homologous ancestral beta-beta-hairpins. This is part of the reason why predicting their three-dimensional (3D) structure, especially by homology modeling, is a major challenge. Recently, DeepMind's AlphaFold v2 (AF2) became the first structure prediction method to reach close-to-experimental atomic accuracy in CASP even for difficult targets. However, membrane proteins, especially OMBBs, were not abundant during its training, raising the question of how accurate the predictions are for these families. In this study, we assessed the performance of AF2 in the prediction of OMBBs of various topologies using an in-house-developed tool for the analysis of OMBB 3D structures, barrOs. In agreement with previous studies on other membrane protein classes, our results indicate that AF2 predicts OMBB structures at high accuracy independently of the use of templates, even for novel topologies absent from the training set. These results provide confidence on the models generated by AF2 and open the door to the structural elucidation of novel OMBB topologies identified in high-throughput OMBB annotation studies.