Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt an off-pathway “deactive” state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here we report the structure of mitochondrial complex I from the thoracic muscles of the model protostomian Drosophila melanogaster . We show that, although D. melanogaster complex I ( Dm -CI) does not deactivate the resting state of Dm -CI adopts multiple conformations. We identify a new helix-locked open state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm -CI structure and conformational states to those observed in bacteria, yeast and mammals provides insight into the roles of subunits across organisms, explains why Dm -CI does not deactivate and reveals incompatibilities with current mechanistic models of complex I turnover. Additionally, the Dm -CI structure and novel regulatory mechanism will allow for the development of more selective pesticides for agriculture and human disease. ### Competing Interest Statement The authors have declared no competing interest.