Respiratory complex I is a well-known player in aerobic respiration; it couples the transfer of two electrons from NADH to ubiquinone alongside with proton pumping. Bacterial complex I is usually composed of fourteen subunits forming three different modules [1]. While the full-size version of the complex is the most commonly found among eubacteria, an 11-subunit version, lacking the NADH-oxidizing module, is surprisingly widely distributed [2]. This version, considered as ancestral [2,3], was initially described in photosynthetic systems where it has been adapted to use ferredoxin as electron donor [4,5]. The eleven genes encoding the complex I-like enzyme was notably detected in the genomes of several representative bacteria performing strictly anaerobic metabolisms, including Desulfitobacterium. This bacterium is a versatile organism particularly known for its ability to respire organohalogens, an energy metabolism that remains poorly understood. Here, we investigated the role of the complex I-like enzyme in the metabolism of Desulfitobacterium hafniense strain DCB-2 by cultivating the bacterium using different combinations of electron donors and acceptors. In order to reveal which energy metabolism relies on a functional complex I-like enzyme, the different cultures were treated with rotenone, a complex I inhibitor. In addition, a tandem mass-tag proteomic approach was developed to compare the abundance of the enzyme in the different growth conditions and to start identifying potential redox partners. In most of the tested conditions, the inhibition of the enzyme resulted in strong growth defects. However, when H2 was used as electron donor for the culture, the inhibition was mostly abolished or no longer observed, independently of the nature of the electron acceptor. These results suggest that the 11-subunit complex I-like enzyme is essential for D. hafniense strain DCB-2 when the electrons are delivered from metabolites located in the cytoplasm. In addition, the proteomic data indicated interesting redox partner candidates to substitute for the missing NADH-oxidizing module, among which two ferredoxins. References 1 Berrisford JM, Baradaran R & Sazanov LA (2016) Structure of bacterial respiratory complex I. Biochim Biophys Acta - Bioenerg 1857, 892–901. 2 Moparthi VK & Hägerhäll C (2011) The Evolution of Respiratory Chain Complex I from a Smaller Last Common Ancestor Consisting of 11 Protein Subunits. J Mol Evol 72, 484–497. 3 Esposti MD (2016) Genome analysis of structure-function relationships in respiratory complex i, an ancient bioenergetic enzyme. Genome Biol Evol 8, 126–147. 4 Schuller JM, Birrell JA, Tanaka H, Konuma T, Wulfhorst H, Cox N, Schuller SK, Thiemann J, Lubitz W, Sétif P, Ikegami T, Engel BD, Kurisu G & Nowaczyk MM (2019) Structural adaptations of photosynthetic complex I enable ferredoxin-dependent electron transfer. Science (80- ) 363, 257–260. 5 Friedrich T & Scheide D (2000) The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases. FEBS Lett 479, 1–5