Deactivation blocks proton pathways in the mitochondrial complex I Michael Röpkea, Daniel Rieplb,1, Patricia Saurab,1, Andrea Di Lucab,1, Max E. Mühlbauera,b, Alexander Jussupowa,b, Ana P. Gamiz-Hernandezb, and Ville R. I. Kailaa,b,2 aDepartment Chemie, Technische Universität München, D-85747 Garching, Germany; and bDepartment of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved May 11, 2021 (received for review September 16, 2020) Cellular respiration is powered by membrane-bound redox en- The recently resolved cryo-electron microscopy (cryoEM) zymes that convert chemical energy into an electrochemical pro- structures of the “deactive” mammalian complex I at around 4-Å ton gradient and drive the energy metabolism. By combining resolution highlighted conformational changes around several large-scale classical and quantum mechanical simulations with subunits close to the interface between the hydrophilic and cryo-electron microscopy data, we resolve here molecular details membrane domains of complex I. Particularly interesting are the of conformational changes linked to proton pumping in the mam- conformational changes around the membrane domain subunits malian complex I. Our data suggest that complex I deactivation ND4L, ND3, and ND6 (ND for NADH Dehydrogenase) that blocks water-mediated proton transfer between a membrane- form a bundle of 11 transmembrane (TM) helices, connected by bound quinone site and proton-pumping modules, decoupling long loop regions (14–16). Notably, it was observed that TM3 of the energy-transduction machinery. We identify a putative gating ND6 transitions from a fully α-helical form in the “active” state region at the interface between membrane domain subunits ND1 to a π-bulge around residues 60 to 65 during the deactivation and ND3/ND4L/ND6 that modulates the proton transfer by confor- process (14–16). Although the exact relevance of these confor- mational changes in transmembrane helices and bulky residues. mational transitions remains debated, it is notable that several The region is perturbed by mutations linked to human mitochon- point mutations in the vicinity of these regions have been linked drial disorders and is suggested to also undergo conformational to mitochondrial disease (11), supporting their possible func- changes during catalysis of simpler complex I variants that lack the tional relevance. Structural changes during complex I deactiva- BIOPHYSICS AND COMPUTATIONAL BIOLOGY “active”-to-“deactive” transition. Our findings suggest that confor- tion, inferred from the lack of density in the cryoEM maps mational changes in transmembrane helices modulate the proton – transfer dynamics by wetting/dewetting transitions and provide (14 16), were also suggested to take place in several loop regions important functional insight into the mammalian respiratory of the membrane domain, near the Q10 binding site tunnel, and complex I. around the supernumerary subunits NDUFA5/NDUFA10 (16, 18). However, the functional consequences of these structural cell respiration | bioenergetics | molecular simulations | QM/MM | cryoEM changes and their coupling to the biological activity still remain unclear. n mitochondrial cellular respiration, the membrane-bound Ienzyme complexes I, III, and IV convert chemical energy in- Significance to a flux of electrons toward dioxygen (1–6). The free energy of the process is transduced by pumping protons across the inner The electron transport chain of mitochondria is initiated by the mitochondrial membrane (IMM), powering oxidative phos- respiratory complex I that converts chemical energy into a phorylation and active transport (7, 8). The electron transport proton motive force to power synthesis of adenosine triphos- process is initiated by the respiratory complex I (NADH:ubi- phate. On a chemical level, complex I catalyzes elementary quinone oxidoreductase), a 45-subunit modular enzyme ma- electron and proton transfer processes that couple across large chinery that shuttles electrons from nicotinamide adenine molecular distances of >300 Å. However, under low oxygen concentrations, the respiratory chain operates in reverse mode dinucleotide (NADH) to ubiquinone (Q10) and transduces the free energy by pumping protons across the IMM, generating a and produces harmful reactive oxygen species. To avoid cell proton motive force (pmf) (1, 4–6) (Fig. 1). This proton-coupled damage, the mitochondrial complex I transitions into a deac- electron transfer reaction is fully reversible, and complex I can tive state that inhibits turnover by molecular principles that also operate in reverse electron transfer (RET) mode, powering remain elusive. By combining large-scale molecular simulations ubiquinol oxidization by consumption of the pmf. Such RET with cryo-electron microscopy data, we show here that com- modes become prevalent under hypoxic or anoxic conditions that plex I deactivation blocks the communication between proton may result, e.g., from stroke or tissue damage (9), during which pumping and redox modules by conformational and hydration the electrons leak from complex I to molecular oxygen and result changes. in the formation of reactive oxygen species (ROS) with physio- – Author contributions: V.R.I.K. designed research; M.R., D.R., P.S., A.D.L., M.E.M., A.J., and logically harmful consequences (9 11). To regulate this poten- A.P.G.-H. performed research; M.R., D.R., P.S., A.D.L., M.E.M., A.J., and A.P.G.-H. contrib- tially dangerous operation mode, the mammalian complex I can uted new reagents/analytic tools; M.R., D.R., P.S., A.D.L., M.E.M., A.J., A.P.G.-H., and V.R.I.K. analyzed data; and V.R.I.K. wrote the paper. transition into a “deactive” (D) state with low Q10-turnover ac- tivity (12, 13). Although some structural changes involved in the The authors declare no competing interest. “active”-to-“deactive” (A/D) transition were recently resolved This article is a PNAS Direct Submission. (14–16), the molecular details of how this transition regulates This open access article is distributed under Creative Commons Attribution-NonCommercial- enzyme turnover and its relevance during in vivo conditions still NoDerivatives License 4.0 (CC BY-NC-ND). remain puzzling. Moreover, it is also debated whether confor- 1D.R., P.S., and A.D.L. contributed equally to this work. mational changes linked to this transition are involved in the 2To whom correspondence may be addressed. Email: [email protected]. native catalytic cycle of all members of the complex I superfamily This article contains supporting information online at https://www.pnas.org/lookup/suppl/ or whether this transition is specific for the mitochondrial en- doi:10.1073/pnas.2019498118/-/DCSupplemental. zyme (12, 13, 17). Published July 16, 2021. PNAS 2021 Vol. 118 No. 29 e2019498118 https://doi.org/10.1073/pnas.2019498118 | 1of10 Downloaded by guest on September 28, 2021 the coupling between proton pumping- and redox-modules upon A complex I deactivation. The explored molecular principles are of general importance for elucidating energy transduction mecha- nisms in the mammalian respiratory complex I and possibly other NADH bioenergetic enzyme complexes, but also for understanding the NAD+ development of mitochondrial diseases. ND2 ND6 ND3 FMN Asp66 Glu34 Glu70 Results TM3ND6 Conformational Dynamics Modulate Water-Mediated Proton Transfer Tyr59 Lys105 Glu34 Reactions. NDUFA5 2e− To probe the dynamics of the mammalian complex I, ND4L ND1 we embedded the 45-subunit “active” state of mouse complex I NDUFA10 (19) in a mitochondrial inner lipid membrane-model with POPC/ POPE/cardiolipin (2:2:1), solvated the model with water mole- + + + + N-side H H H H cules and 150 mM NaCl, and simulated the ca. 1 million atom system in total for around 8.5 μs using atomistic MD simulations. QH2 A quinone or quinol molecule was modeled in the primary binding Q site near the N2 iron–sulfur center (20) or in a membrane-bound ND3 ND1 binding site located near the Q-tunnel kink region at ND1 that ND2 ND4L ND6 P-side ND5 ND4 was recently predicted based on simulations and validated exper- imentally (19, 21). Simulations were also performed with the SI Appendix TM3 ND3 Q-cavity left in an empty (apo) state (refer to ,Fig.S1 B C and Table S1 for simulation setups and Table S2 for modeled active state protonation states). To obtain molecular insight into the “deac- model tive” state, we constructed an intact atomistic model of this form ND6 by targeting the “active” state model toward the experimental TM3 ND6 ND4L TM4 cryoEM “deactive” density (EMD: 4356) (16) using an MD flex- TMTM44 deactive state Tyr69r69 ible fitting (MDFF) approach, followed by unrestrained MD cryoEM map D simulation for a total simulation time of ca. 6 μs (Fig. 1B). In the MDFF Tyr69ND6 Phe67Phe67 targeted MDFF simulations, the cryoEM density acts as an ex- acactivetive sstatetate ternal biasing potential that guides the dynamics of the residues, deactive state deactive state whereas for unresolved regions, the dynamics is directed only by model the biomolecular force field (22). This modeling approach is likely Phe67ND6 to provide a more balanced description of the “deactive” state and a better comparison to our “active” state simulations, as the E former state could not be experimentally resolved at the same “ ” “ 150 atomic level of detail as the active state. The obtained deac- S8 S12 6g2j ” “ ” active tive model, created from our active state simulation setup, 125 6ztq S1 S13 resembles the previously refined 3.9-Å structure of the former S2 S9 100 [Protein Data Bank (PDB) ID: 6G72] (16) for the experimentally (°) 75 resolved regions but contains more atomic detail for unresolved Φ S6 S11 parts (Fig. 1 C and D and SI Appendix, Figs. S2, S3, and S12, 50 6g72 Materials and Methods). The global dynamics observed in the MD S15 deactive 25 S10 simulations resemble the dynamics inferred from the local reso- S14 0 S7 lution of the cryoEM map (SI Appendix,Fig.S2A), including a 0 200 400 600 800 1000 Time (ns) relative twist of the hydrophilic domain relative to the membrane domain experimentally described before (14–16, 23) (SI Appendix, Fig.