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  1. eLife assessment

    This important work provides new insights into the structure and function of respiratory complex I. The cryoEM data are convincing but the assignment of different conformations of the enzyme complex to specific functional states has not yet been conclusively determined. This work will be of interest to researchers studying the molecular basis of energy metabolism, the evolution of respiratory enzyme complexes, and mitochondrial diseases.

  2. Reviewer #1 (Public Review):

    Cryo-EM structures of respiratory complex I have in recent years have a large impact on our understanding of its mechanism, regulation, assembly, and evolution. However, the coupling mechanism of complex I is still not clear, and controversies exist about whether certain conformations are part of the catalytic cycle or arise from the deactivation of the enzyme. Padavannil and colleagues now add to the story with the first structures of insect complex I, from the model organism Drosophila melanogaster. One of the rationales for choosing this organism is that it lacks the active-to-deactive (A-to-D) transition that prevents the enzyme from going in reverse, which should make the interpretation of any different conformations more straightforward.

    The authors showed that the A-D transition seen in mammals and fungi was indeed not present in Drosophila complex I and they determined the cryo-EM structure. In contrast to especially mammalian complex I, which is often found in an "open" and a "closed" state, there was only a single conformation. Drosophila complex I has lost two accessory subunits compared to the mammalian complex, and several other subunits have lost or gained elements, with possible implications for the assembly, stability, or regulation of the complex. The interface of the two peripheral and membrane arms was poorly resolved. A focused classification on this region yielded distinct structures, differing in the angle of the two arms and in the presence or absence of an alpha helix at the N terminus of subunit NDUFS4 (the "lock helix"), a region that is not present in mammalian or yeast complex I. The authors observe a transition between two states named "closed" and "locked open" and speculate that the transition constitutes a deactivation mechanism in insect complex I.

    The conclusions of the paper are for the most part solid and supported by the data. Only the interpretation of the significance of the "lock helix" is not convincing: without any evidence, it is assumed to be a regulatory element responsible for an off-pathway deactive state. The nomenclature "closed" and "locked open" is unfortunate, as most of the structural features that differ between the states are reversed compared to the mammalian closed and open states: the disorder of several loops in the quinone binding regions and the presence of absence of a π bulge in helix 4 of the ND6 subunit. Thus, the "locked open" state, which the authors assign as an off-pathway resting state, shares the features of the mammalian closed state, which in all catalysis models is considered an "active" state. An especially important feature in the closed state is the alpha-helical conformation of ND6-helix 4, which has been shown to support a water wire connection from the Q site to the membrane arm, suggesting a role in proton transfer. Conversely, all structures considered as possible D states in mammalian or yeast complex I are open and show disordered loops and a π bulge. These features as shared by the "closed" state of Drosophila, which is however assumed to be on-pathway.

  3. Reviewer #2 (Public Review):

    In this work, the authors present a high-resolution cryo-EM structure of mitochondrial complex I, which was isolated from the model protostomian Drosophila melanogaster. Although multiple structures of related complexes have been published earlier, this system is particularly interesting as it seems not to adopt a so-called off-pathway "deactive" (D) state in contrast to the complex from Deuterostomia (including mammals) and therefore may provide novel mechanistic insights into complex I. The work is interesting, as it provides a novel contribution to the current discussion about the assignment of structural conformations to states in the catalytic cycle and/or in the active/deactive state transition of the complex.

  4. Reviewer #3 (Public Review):

    This paper by Padavannil et al. presents a new cryo-EM structure of mitochondrial complex I from Drosophila melanogaster. This is a timely and important study - the new structure and comparative analysis would allow new insights into mitochondrial complex I mechanism and regulation. The major strength is the advanced CryoEM analysis and structure resolution. The manuscript is well-written and scientifically sound, but a clear weakness is the lack of classical enzyme kinetic analysis of the A/D transition, even though this is supposed to be the foundation for the main conclusion of the manuscript. However, the interpretation of the data is rational and scientifically justified.