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Respiratory complex I — structure, mechanism and evolution
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ScienceDirect Respiratory complex I — structure, mechanism and evolution Kristian Parey1,2,3,5, Christophe Wirth4,5, Janet Vonck2 and Volker Zickermann1,3 Respiratory complex I is an intricate multi-subunit membrane protein with a central function in aerobic energy metabolism. During the last years, structures of mitochondrial complex I and respiratory supercomplexes were determined by cryo-EM at increasing resolution. Structural and computational studies have shed light on the dynamics of proton translocation pathways, the interaction of complex I with lipids and the unusual access pathway of ubiquinone to the active site. Recent advances in understanding complex I function include characterization of specific conformational changes that are critical for proton pumping. Cryo-EM structures of the NADH dehydrogenase-like (NDH) complex of photosynthesis and a bacterial membrane bound hydrogenase (MBH) have provided a broader perspective on the complex I superfamily. Addresses 1 Institute of Biochemistry II, University Hospital, Goethe University, Frankfurt am Main, Germany 2
Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 3 Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 4 Institute of Biochemistry and Molecular Biology, ZBMZ, Medical Faculty, Albert-Ludwigs-University Freiburg, Freiburg, Germany Corresponding authors: Vonck, Janet ([email protected]), Zickermann, Volker ([email protected]) 5 Contributed equally. Current Opinion in Structural Biology 2020, 63:1–9 This review comes from a themed issue on Membranes
the inner membrane of mitochondria or the cell membrane of bacteria [1,2]. The reaction is tightly coupled and reversible. Complex I dysfunction is associated with numerous neuromuscular or neurodegenerative diseases in humans [3,4]. Under conditions that promote reverse electron flow, complex I is a major source of deleterious reactive oxygen species (ROS) [5]. The reversible active (A) to deactive (D) transition [6] is thought to protect against excessive ROS formation [7]. Complex I is one of the largest membrane protein complexes. It is L-shaped with a peripheral arm involved in electron transfer from NADH to Q and a membrane arm responsible for proton pumping (Figure 1). It consists of 14 central subunits, 7 in the peripheral arm and 7 in the membrane arm, that are conserved from bacteria to humans, and about 30 accessory subunits that occur almost exclusively in eukaryotic complex I. We will here use the subunit designations for human complex I throughout. The structures of respiratory complex I from bacteria and from the aerobic yeast Yarrowia lipolytica were solved by X-ray crystallography [8–10]. Since the resolution revolution in cryo-EM [11], structures of mammalian and Y. lipolytica complex I and respiratory supercomplexes were determined by this technique at increasing resolution [12–15,16,17,18,19,20,21]. High-resolution structures have enabled molecular modeling and molecular dynamics simulation approaches that will be indispensable for unravelling the mechanism of redox-linked proton translocation [22,23].
Edited by Beili Wu and Fei Sun
https://doi.org/10.1016/j.sbi.2020.01.004
In this review we focus on progress in structure determination, functional studies and molecular modelling of complex I, its supercomplexes and related structures over the past three years.
0959-440X/ã 2020 Elsevier Ltd. All rights reserved.
Substrate binding sites
Introduction In aerobic energy metabolism, ATP is largely provided by oxidative phosphorylation. The redox-driven proton translocating complexes of the respiratory chain generate the proton motive force that drives ATP synthase. Respiratory complex I (proton pumping NADH:ubiquinone oxidoreductase) couples electron transfer from NADH to ubiquinone (Q) with translocation of four protons across www.sciencedirect.com
The initial electron acceptor in complex I is a flavin mononucleotide (FMN) in subunit NDUFV1 (Figure 1). Binding of substrate close to FMN was shown by X-ray crystallography and cryo-EM [20,24]. A chain of seven FeS clusters connects the NADH binding site with the Q reduction site (Figure 1) [25]. Cluster N1a in NDUFV2 is thought to have a special function because it is located on the distal side of FMN (Figure 1). Based on a series of high-resolution structures of a NDUFV1/2 fragment, a recent study suggests that the redox-state of N1a controls access of NADH and thus protects against overreduction of complex I [26]. Current Opinion in Structural Biology 2020, 63:1–9
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Figure 1
The topology of the Q reduction site in complex I is remarkably different from that of Q reactive sites in other membrane protein complexes. FeS cluster N2, the immediate reductant for Q, is buried in the peripheral arm above the membrane surface (Figure 1) [27,28]. Hydrophobic Q has to move in a tunnel from the lipid bilayer to the Q reduction site (Figure 2a) [9,10]. Two recent cryoEM maps showed that in the region around the tunnel entrance, subunits of the hydrophilic peripheral arm bind lipid head groups, and lipid tails wrap around the hydrophobic surfaces of amphipathic helices lining the membrane/peripheral arm interface region (Figure 2b) [18,21]. This unusual lipid-protein arrangement is thought to cause thinning of the lipid bilayer to facilitate entry of Q into the tunnel and flipping of the Q head group for fast alternating access to Q reactive sites in complexes I and III (Figure 3b) [21]. In the active site, the Q head group is bound in electron transfer distance to cluster N2 by a tyrosine and a histidine residue (Figure 2a) [9,29]. The long isoprenoid tail is thought to be important for guiding and facilitating movement of Q in the extended access pathway [30]. Computational approaches identified multiple intermediary binding sites in the Q tunnel (Figure 2a) [31,32,33]. In [31], site 1 corresponds to the binding position in the active site described above. Site 1matches the binding site for a Q antagonistic inhibitor identified by X-ray crystallography [10]. Site 2 is close to a bend of the tunnel that is surrounded by a remarkable network of charged residues. This network represents the origin of the so-called hydrophilic axis [9] extending towards the proton pumps. Sitedirected mutagenesis and simulations identified critical residues in a loop of NDUFS7 lining the Q access pathway and the interface of the Q and P modules [34]. Recently, cryo-EM showed binding of a native Q molecule to site 2 in Y. lipolytica complex I [21] that might represent a substrate molecule on its way between
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Q-module N-side matrix P-side IMS P-module Current Opinion in Structural Biology
Overall structure and schematic representation of respiratory complex I. (a) Cryo-EM density map of Y. lipolytica complex I (EMD-4873) with the densities of the distinct subunits in different colors. (b) Cryo-EM structure of Y. lipolytica complex I (PDB ID: 6RFR). Central subunits are labelled, the position of FMN (NADH oxidation site) and of FeS clusters Current Opinion in Structural Biology 2020, 63:1–9
N2 (Q reduction site) are highlighted; FMN and FeS clusters are shown in the inset (assignment of EPR signatures N1 to N5 according to Roessler et al. [66]); a native Q molecule is bound in the access pathway [21]. Accessory subunits are shown transparent. Further notable cofactors are represented: the phosphopantetheine cofactor of acyl carrier proteins with appended fatty acid (PP) [67], the NADPH molecule in NDUFA9 and the zinc ion (Zn) in NDUFS6. (c) The central subunits are assigned to functional modules for NADH oxidation (N module), ubiquinone reduction (Q module) and proton translocation (P module). The N and Q modules form the so-called matrix or peripheral arm, the P module corresponds to the membrane arm. Electrons are transferred from FMN to the Q reduction site by a chain of seven FeS clusters. The three antiporter-like subunits ND2 (purple), ND4 (cyan), and ND5 (deep blue) harbor protontranslocation pathways each consisting of two half-channels connecting to the N-side (mitochondrial matrix) and the P-side (intermembrane space, IMS), respectively; the position of a putative fourth proton channel is discussed. Each antiporter-like subunit contains two discontinuous helices (TMH 7a/b, TMH 12a/b), a central lysine residue in the center of the H+ translocation pathway, and a Lys-Glu pair between helices 7 and 5 implicated in lateral signal transfer. The protonation state of critical residues is thought to control the dynamic hydration and thus connectivity of half channels [23,38,39]. Switching between a p-bulge and a regular a-helix was observed for TMH3 of ND6 (orange) [18,44]. www.sciencedirect.com
Structure and function of respiratory complex I Parey et al.
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Figure 2
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Q tunnel and lipids. (a) The Q tunnel (gray) in complex I from Y. lipolytica (PDB ID: 6RFR) was calculated using CAVER [68]. Free energy calculations and simulations identified multiple intermediate binding sites in the Q tunnel [31,32,33]; structural evidence for binding of Q to positions matching sites 1 [9], 10 [10,20], and 2 [21] was provided by X-ray crystallography and cryo-EM. The Q head group in site 1 was fitted manually according to Figure 4 of Ref. [9]. (b) A total of 33 phospholipids were modelled in the density map of complex I from Y. lipolytica [21]. For orientation, the Q molecule in site 2 is shown in blue. Lipid head groups in the interface region are shifted towards a plateau above the membrane surface level defined by lipid head groups in the rest of the membrane arm; the lower panel shows a view from the backside of complex I. In the interface region lipid tails are bent and wrapped around the hydrophobic surface of amphipathic helices [21]. Figure 3
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The respirasome (supercomplex I1III2IV1). (a) Cryo-EM map of the respirasome from sheep (EMD-8130) [14]; side view (upper) and view from the matrix (lower), complex I (orange), dimer of complex III (red), complex IV (green). In the lower panel, accessory complex I subunit NDUFA11 providing contact in the membrane plane is highlighted in magenta. (b) Schematic representation of the respirasome with cofactors highlighting the electron (solid arrows) and substrate transfer (dotted arrows) in the supercomplex; asymmetry observed in Q binding to complex III suggests that only the Qo site proximal to complex I is active [44]. Electrons can, however, also be transferred to the Qi site of the other monomer (light blue arrows). Mobility of the Rieske FeS protein (ISP) is critical for bifurcation of electrons at the Qo site; in a bovine respirasome, density indicating rigid protein structure was visible for the distal but not for the proximal ISP, suggesting that the proximal site is active while the distal site is in resting state [53]. www.sciencedirect.com
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the membrane and the active site (Figure 2a). Alternatively, a bound Q might act as a mobile electron carrier shuttling electrons from the Q reduction site to the membrane Q pool [35]. The mechanistic implications of a shuttling Q and electron transfer at or close to site 2 were discussed [32,36]. However, the functional significance of the observed Q binding site remains unresolved and has to be evaluated in future work.
Dynamic water chains and long-range energy transfer in the membrane arm In complex I, the reduction of Q triggers the translocation of protons across the membrane, remote from the Q binding site. The three largest membrane arm subunits ND2, ND4 and ND5 have a common core fold [8] and are related to subunits of Mrp-type sodium proton antiporters [37]. In each subunit, TMH7 and TMH12 are discontinuous (Figure 1c) [8]. Proton-translocation pathways extend along TMH segments 7b and 12b and connect the N and the P side with the central membrane plane [8–10] (Figure 1c). The position of a fourth proton translocation pathway is debated. Recent computational work suggests that channel hydration is dynamic and dependent on the charge state of key residues positioned at the origin of each half channel (Figure 1c) [38,39,40]. These residues line up along the hydrophilic axis that forms a continuous connection of protonatable residues to the Q reduction module (Figure 1c). Q reduction is thought to drive initial proton uptake in ND1 [41], which in turn triggers a signal along the hydrophilic axis towards ND5 [23]. The signal is conveyed by a series of lateral proton transfers and switching of a LysGlu ion pair in each antiporter-like subunit. It controls the concerted formation and decay of water wires and drives vectorial proton movements while back leak reactions are prevented [23]. The described mechanism is still hypothetical but recent structural work identified conformational alterations in TMH8 of the antiporter-like subunits that might reflect changes linked with signal propagation in the hydrophilic axis [18].
form shows a localized unfolding of protein structure around the Q reduction site [12,18,19]. Restructuring during transition into the A form is thought to occur by an induced fit mechanism with Q acting as a template [19]. In the membrane arm, a p-bulge in TMH3 of ND6 is exclusively present in the D form [18]. The interconversion into an extended helix present in the A form is associated with a rotation of the C-terminal helix segment, which becomes visible as a prominent change of side chain positions of two aromatic residues [18]. Interestingly, the p-bulge of TMH3 of ND6 is connected to charged residues of the E channel and is therefore an element of a putative proton translocation pathway (Figure 1c) [18]. The extensive conformational rearrangements in the Q site and in ND6 were not observed in complex I from Y. lipolytica [20]. This is consistent with a lower energy barrier for the transition between A and D form in the yeast enzyme complex [42]. The absence of extensive unfolding around the Q site might be explained by the presence of a Q molecule in site 2 [21], which was not observed in other complex I structures so far.
Structures of complex I in different functional states
A recent study of ovine I1III2 supercomplex showed the presence of two major complex I classes called open and closed state in one data set [44]. The closed state largely matches the A form of bovine and mouse complex I (see above); the open state represented an ensemble of states and showed extensive similarity with the D form. However, biochemical analysis indicated the absence of detectable amounts of the D form in the ovine supercomplex preparation. Based on this finding the authors of this study concluded that the conversion between open and closed state occurs while complex I is in the A form and is therefore part of the catalytic cycle [44]. They suggest that the remarkable rotation of the TMH3 segment of ND6 is an efficient element of conformational coupling within the E pathway [44]. The D form is proposed to represent one of the open-like states with more extensive unfolding of loops around the Q site [44]. Although this study provides exciting new insights, it has to be taken with a grain of salt as the data for evaluating the relative amounts of A and D form in the preparation were not shown.
Complex I from vertebrates and fungi exists either in the A or D form [7,42]. The A form is able to catalyze fast electron transfer to Q. When substrates sustaining complex I turnover are depleted, e.g. during ischemia, complex I spontaneously switches to the resting D form. Return to the A form occurs by slow activating turnovers. This process is impeded or blocked by divalent cations, alkaline pH or covalent modification of a specific cysteine residue in subunit ND3 [43]. Modulation of the A/D transition by thiol reactive agents offers new strategies for mitigating tissue damage in myocardial infarction [5]. The structural basis of the A/D transition was investigated by cryo-EM in bovine and mouse complex I [12,18,19]. The A and D form are related by slight opposing rotations of the matrix arm and a large sector of the membrane arm [12]. The D
The structure of complex I from Y. lipolytica was analyzed by cryo-EM in the deactive state and after freezing the enzyme under turnover [20]. Several lines of evidence indicate that electron transfer from N2 to Q occurs while the Q head group is bound to a tyrosine and a histidine residue close to cluster N2 [9,29]. However, in the cryoEM structure under turnover, a Q molecule was modelled in a different position between the tip of the b1b2 loop and helix 2 of NDUFS7 [20]. The Q position observed overlaps with the binding site of an inhibitor determined by X-ray crystallography [10] and matches one of the sites identified in molecular simulations (site 1) (Figure 2a) [31]. These data support a two-state stabilization change mechanism [45] that postulates cycling between two Q
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Structure and function of respiratory complex I Parey et al.
binding positions permitting either electron transfer (E state) or protonation steps (P state) in the reduction of Q. The rearrangement of the Q binding site associated with stabilization of negatively charged Q intermediates is thought to generate the power stroke that is transmitted to the membrane domain to drive the proton pumps [20,45]. The structural data also concur with free energy calculations predicting that after formation in site 1, QH2 relaxes to site 1[31].
Identification of mobile loop elements at the membrane/matrix arm interface We have proposed that the redox chemistry of Q drives the concerted rearrangement of three loops in NDUFS2, ND3 and ND1 located in and around the Q reduction site [10,20]. The NDUFS2 loop harbors the conserved histidine that binds the Q head group in the active site. Modelling in different conformations in a variety of structures indicates flexibility of the loop. The nearby TMH5-6 loop of ND1 carries several conserved acidic residues and represents the endpoint of the hydrophilic axis connecting to the proton pumps. The ND3 loop carries a cysteine residue that is employed as a reporter group for the A/D transition because it shows selective accessibility, for example, for NEM labeling, in the D form only [43]. Introducing a cysteine residue in helix 2 of NDUFS7 by site-directed mutagenesis permitted formation of a disulfide bond with the ND3 cysteine [46]. Electron transfer was essentially unaffected but proton pumping stopped after formation of the disulfide bond, indicating that unrestricted movement of the ND3 loop is essential for the coupling mechanism [46].
Structure of a complex I assembly intermediate Complex I assembly is a convoluted process that is supported by assembly factors [47,48]. Assembly factor NDUFAF2 has been associated with attachment of the N module to nascent complex I [49]. Deletion of the gene for accessory subunit NDUFS6 in Y. lipolytica stalled complex I assembly and blocked release of NDUFAF2 [50]. The cryo-EM structure of the assembly intermediate was recently determined and offered clues on the interplay of NDUFAF2 with accessory subunits NDUFS4, NDUFS6 and NDUFA12 in the last step of complex I assembly [21].
Supercomplexes Respiratory supercomplexes were initially isolated by blue-native PAGE after mild solubilization of mitochondrial membranes by digitonin [51], and later visualized by single-particle electron microscopy [52]. In the last few years cryo-EM structures have been determined from several mammalian species (Figure 3a) [14,15,16,17,44,53]. Most of these structures have the composition I1III2IV1, the so-called respirasome, that together with Q and cyt c contains all components www.sciencedirect.com
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to transfer electrons from NADH to oxygen (Figure 3b). The basic architecture of the respirasome is the same in all species, with complex III nestled at the concave side of the complex I membrane arm and complex IV attached at the distal end. Intercomplex contacts are established by accessory subunits, most notably NDUFA11 between complex I and III (Figure 3a). The supercomplex structures are usually not very rigid and in some studies multiple conformations are found [14,53]. A cryo-electron tomography study [54] showed that the in vivo arrangement of complexes I and III is conserved not only in mammals, but also in yeast (Y. lipolytica) and plant mitochondria, but that the position of complex IV is variable between and within species. The supercomplex structures show that there is no intraprotein path for Q between complexes I and III and experimental work supports the availability of reduced Q outside the supercomplex [55,56]. These data provide strong evidence against substrate channelling. However, one of the Q binding sites of complex III faces complex I, leading to a short diffusion pathway, and two cryo-EM studies have found evidence for asymmetry in complex III that supports preferential Q entry at this site (Figure 3) [44,53]. There is no evidence that supercomplex formation would play a role in ROS production [57]. A recent hypothesis states that supercomplexes have evolved to ensure a homogeneous distribution of complexes in the crowded environment of the mitochondrial inner membrane to promote an efficient and rapid catalysis of respiration [58].
Complex I superfamily Complex I is a member of a superfamily of membranebound redox enzymes that have adapted ancient modules to cope with diverse evolutionary pressures [59,60]. Recently, structures of some of these complexes have been determined by cryo-EM, providing a structural basis for the evolutionary relationships in this family. Cyanobacteria are the organisms that first evolved oxygenic photosynthesis. They contain several enzymes from the complex I superfamily with diverse functions, including respiration, cyclic electron flow and CO2 acquisition [61]. The cyanobacterial NDH-1L complex is the evolutionary origin of the NADH dehydrogenase-like (NDH) complex in chloroplasts. This ‘photosynthetic complex I’ should not be confused with respiratory complex I of plant mitochondria [62]. Two cryo-EM studies of NDH1L from Thermosynechococcus elongatus [63,64] now shed light on its structure (Figure 4b). The NDH complex is involved in cyclic electron flow, transferring electrons from ferredoxin, reduced by photosystem-1, to plastoquinone and pumping protons across the thylakoid membrane, thereby adjusting the ATP/NADPH ratio produced by photosynthesis. It shares 11 of the 14 core subunits of respiratory complex I, only lacking the three subunits of the N module that transfer electrons from Current Opinion in Structural Biology 2020, 63:1–9
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Figure 4
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Conserved structural and functional elements in complex I and related complexes. Structures and schematic representations of (a) MBH complex from P. furiosus (PDB ID: 6CFW, EMD-7468) (b) NDH complex from T. elongatus (PDB ID: 6HUM EMD-0281) (c) bacterial complex I from T. thermophilus (PDB ID: 4HEA) and (d) mitochondrial complex I from mouse (PDB ID: 6G2J, EMD-4345); coloring of central subunits reflects evolutionary relations between the different subunits of the complexes (note that MbhI is related to ND3 and ND5). Accessory subunits are shown in gray. The loop cluster at the interface of Q and P module in complex I is conserved in NDH and MBH. Movement of the ND3 loop (yellow) was shown to be essential for coupling Q reduction to proton translocation [46].
NADH to the Q binding site (Figure 4b). Instead, four oxygenic-photosynthesis-specific subunits facilitate electron transfer from ferredoxin to plastoquinone. Some anaerobic archaea have a hydrogen gas-evolving membrane-bound hydrogenase (MBH) that uses ferredoxin as electron donor to reduce protons to molecular hydrogen [60]. MBH is a sodium pump, generating a Current Opinion in Structural Biology 2020, 63:1–9
sodium gradient that is used to energise a sodium-driven ATP synthase. The cryo-EM structure of the 14-subunit, 300 kDa MBH from Pyrococcus furiosus [65] (Figure 4a) shows a proton-translocating unit similar to the ND2/ ND6/ND4L unit of complex I (Figure 4c). A putative four-subunit sodium-translocating module with homology to Mrp H+/Na+ antiporter subunits is located at the distal end of the membrane domain. This topology www.sciencedirect.com
Structure and function of respiratory complex I Parey et al.
suggests that the redox reaction is initially coupled to generation of a proton gradient, which in turn drives Na+ translocation [65]. Interestingly, the membrane anchored hydrogenase module, consisting of four orthologs of the complex I Q module and an ortholog of ND1, is attached to the opposite end of the proton-pumping unit compared to complex I (Figure 4a,c,d). MBH has a functional, H2 evolving [NiFe] hydrogenase unit, MbhL and MbhJ with a NiFe center and an FeS cluster, respectively, while the related subunits (NDUFS2 and NDUFS7) in complex I have lost the NiFe center and are involved in Q reduction. This supports the proposed evolution of complex I from an H2-evolving ancestor [60]. Intriguingly, loops critical for coupling of Q reduction to proton pumping already have equivalents in MBH, suggesting a conservation of the coupling mechanism despite extensive differences in the redox-chemistry.
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Kotlyar AB, Vinogradov AD: Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase. Biochim Biophys Acta 1990, 1019:151-158.
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Dro¨se S, Stepanova A, Galkin A: Ischemic A/D transition of mitochondrial complex I and its role in ROS generation. Biochim Biophys Acta 2016, 1857:946-957.
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Efremov RG, Sazanov LA: Structure of the membrane domain of respiratory complex I. Nature 2011, 476:414-420.
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Conclusions
11. Ku¨hlbrandt W: The resolution revolution. Science 2014, 343:1443-1444.
The revolution in cryo-EM technology triggered tremendous progress in understanding the structure of mitochondrial complex I and respiratory supercomplexes. Structural characterization of complex I in different functional states is now an area of intense research and molecular simulation approaches and functional studies have provided exciting new insights into the inner workings of this intricate molecular machine. Common structural features in evolutionary related enzyme complexes offer clues on a universal coupling mechanism but also identify specific adaptations within the complex I superfamily. Despite this amazing gain of knowledge, consensus on the molecular mechanism of redox-linked proton translocation is still lacking. The combination of different experimental approaches is needed to obtain a consistent and comprehensive picture in the future.
Conflict of interest statement Nothing declared.
Acknowledgements This work was supported by the German Research Foundation (ZI 552/4-2 and ZI 552/5-1 to V.Z.) and by the Max Planck Society.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Agip AA, Blaza JN, Fedor JG, Hirst J: Mammalian respiratory complex I through the lens of cryo-EM. Annu Rev Biophys 2019, 48:165-184.
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Sazanov LA: A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat Rev Mol Cell Biol 2015, 16:375-388.
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Rodenburg RJ: Mitochondrial complex I-linked disease. Biochim Biophys Acta 2016, 1857:938-945.
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10. Zickermann V, Wirth C, Nasiri H, Siegmund K, Schwalbe H, Hunte C, Brandt U: Mechanistic insight from the crystal structure of mitochondrial complex I. Science 2015, 347:44-49.
12. Zhu J, Vinothkumar KR, Hirst J: Structure of mammalian respiratory complex I. Nature 2016, 536:354-358. 13. Fiedorczuk K, Letts JA, Degliesposti G, Kaszuba K, Skehel M, Sazanov LA: Atomic structure of the entire mammalian mitochondrial complex I. Nature 2016, 538:406-410. 14. Letts JA, Fiedorczuk K, Sazanov LA: The architecture of respiratory supercomplexes. Nature 2016, 537:644-648. 15. Gu J, Wu M, Guo R, Yan K, Lei J, Gao N, Yang M: The architecture of the mammalian respirasome. Nature 2016, 537:639-643. 16. Guo R, Zong S, Wu M, Gu J, Yang M: Architecture of human mitochondrial respiratory megacomplex I2III2IV2. Cell 2017, 170:1247-1257 Low resolution architecture of the respiratory megacomplex I2III2IV2 and 3.9 A˚ resolution structure of the I1III2IV1 supercomplex from human. Subregion refinements of the latter result in structures of human complex I peripheral and membrane arms at 3.4–3.7 A˚ resolution, respectively. 17. Wu M, Gu J, Guo R, Huang Y, Yang M: Structure of mammalian respiratory supercomplex I1III2IV1. Cell 2016, 167:1598-1609. 18. Agip AA, Blaza JN, Bridges HR, Viscomi C, Rawson S, Muench SP, Hirst J: Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states. Nat Struct Mol Biol 2018, 25:547-556 This study compares the structures of mouse complex I in its active form at 3.3 A˚ resolution, with the structure in the deactive form at 3.9 A˚ resolution. It provides a detailed model for the A/D transition mechanism by comparing the structures further with bacterial complex I, and in particular, identifies conformational changes at key elements occurring during activation. 19. Blaza JN, Vinothkumar KR, Hirst J: Structure of the deactive state of mammalian respiratory complex I. Structure 2018, 26:312-319 This study shows that the deactive form of bovine complex I is chararcterized by localized unfolding around the Q binding site. 20. Parey K, Brandt U, Xie H, Mills DJ, Siegmund K, Vonck J, Ku¨hlbrandt W, Zickermann V: Cryo-EM structure of respiratory complex I at work. eLife 2018, 7:e39213 Structure of complex I from Yarrowia lipolytica in the deactive state and after capturing the enzyme during turnover. 21. Parey K, Haapanen O, Sharma V, Ko¨feler H, Zu¨llig T, Prinz S, Wittig I, Siegmund K, Mills DJ, Vonck J et al.: High-resolution cryo-EM structures of respiratory complex I - mechanism, assembly and disease. Sci Adv 2019, 5:eaax9484 The cryo-EM structure of complex I from Yarrowia lipolytica at 3.2 A˚ resolution identifies a bound Q molecule and an unusal lipid-protein arrangement at the interface of membrane and matrix arm. The structure of a complex I mutant lacking accessory subunit NDUFS4 and the Current Opinion in Structural Biology 2020, 63:1–9
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structure of an assembly intermediate provide molecular insights into the cause of complex I-linked Leigh syndrome and into complex I assembly. 22. Haapanen O, Sharma V: A modeling and simulation perspective on the mechanism and function of respiratory complex I. Biochim Biophys Acta 2018, 1859:510-523. 23. Kaila VRI: Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I. J R Soc Interface 2018, 15:20170916 This review article presents a detailed mechanistic model for redox-linked proton translocation in complex I. 24. Berrisford JM, Sazanov LA: Structural basis for the mechanism of respiratory complex I. J Biol Chem 2009, 284:29773-29783. 25. Sazanov LA, Hinchliffe P: Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 2006, 311:1430-1436. 26. Schulte M, Frick K, Gnandt E, Jurkovic S, Burschel S, Labatzke R, Aierstock K, Fiegen D, Wohlwend D, Gerhardt S et al.: A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I. Nat Commun 2019, 10:2551 This study describes several high resolution structures of the electron input domain and provides evidence for a molecular switch that prevents overreduction of complex I. 27. Efremov RG, Baradaran R, Sazanov LA: The architecture of respiratory complex I. Nature 2010, 465:441-445. 28. Hunte C, Zickermann V, Brandt U: Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 2010, 329:448-451. 29. Tocilescu MA, Fendel U, Zwicker K, Dro¨se S, Kerscher S, Brandt U: The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction. Biochim Biophys Acta 2010, 1797:625-632. 30. Fedor JG, Jones AJY, Di Luca A, Kaila VRI, Hirst J: Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I. Proc Natl Acad Sci U S A 2017, 114:12737-12742 Study investigating the kinetics of complex I with Q ranging from Q1 to Q10 and offering clues on the Q access pathway and the importance of the long isoprenoid chain of the physiological substrate. 31. Warnau J, Sharma V, Gamiz-Hernandez AP, Di Luca A, Haapanen O, Vattulainen I, Wikstro¨m M, Hummer G, Kaila VRI: Redox-coupled quinone dynamics in the respiratory complex I. Proc Natl Acad Sci U S A 2018, 115:E8413-E8420 This in silico study identifies intermediate Q binding sites within the extended Q cavity and shows that the dynamics of Q is redox-state dependent. 32. Haapanen O, Djurabekova A, Sharma V: Role of second quinone binding site in proton pumping by respiratory complex I. Front Chem 2019, 7:221 http://dx.doi.org/10.3389/fchem.2019.00221 Molecular dynamics simulations suggesting that the second Q binding site, remote from cluster N2, could play a key role in the catalytic cycle. 33. Teixeira MH, Arantes GM: Balanced internal hydration discriminates substrate binding to respiratory complex I. Biochim Biophys Acta 2019, 1860:541-548 This study uses computational approaches to understand the impact of hydration of the unusual quinone binding cavity in complex I and isoprenoid chain length of quinone. 34. Galemou Yoga E, Haapanen O, Wittig I, Siegmund K, Sharma V, Zickermann V: Mutations in a conserved loop in the PSST subunit of respiratory complex I affect ubiquinone binding and dynamics. Biochim Biophys Acta 2019, 1860:573-581 This study combines site-directed mutagenesis and computational approaches to assess the function of a loop in NDUFS7 for access of Q and coupling of Q reduction to proton pumping.
37. Mathiesen C, Ha¨gerhall C: Transmembrane topology of the NuoL, M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters. Biochim Biophys Acta 2002, 1556:121-132. 38. Di Luca A, Gamiz-Hernandez AP, Kaila VRI: Symmetry-related proton transfer pathways in respiratory complex I. Proc Natl Acad Sci U S A 2017, 114:E6314-E6321 Computational study identifying dynamic water chains for translocating protons across the membrane. 39. Haapanen O, Sharma V: Role of water and protein dynamics in proton pumping by respiratory complex I. Sci Rep 2017, 7:7747 In silico analysis showing the importance of the hydration within the interior of complex I and in particular within the proton translocation pathways as well as in the Q cavity. 40. Kaila VR, Wikstro¨m M, Hummer G: Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I. Proc Natl Acad Sci U S A 2014, 111:69886993. 41. Sharma V, Belevich G, Gamiz-Hernandez AP, Rog T, Vattulainen I, Verkhovskaya ML, Wikstrom M, Hummer G, Kaila VR: Redoxinduced activation of the proton pump in the respiratory complex I. Proc Natl Acad Sci U S A 2015, 112:11571-11576. 42. Maklashina E, Kotlyar AB, Cecchini G: Active/de-active transition of respiratory complex I in bacteria, fungi, and animals. Biochim Biophys Acta 2003, 1606:95-103. 43. Galkin A, Meyer B, Wittig I, Karas M, Scha¨gger H, Vinogradov A, Brandt U: Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I. J Biol Chem 2008, 283:20907-20913. 44. Letts JA, Fiedorczuk K, Degliesposti G, Skehel M, Sazanov LA: Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk. Mol Cell 2019, 75:1131-1146.e6 Investigates the functional significance of supercomplex formation and identifies multiple distinct substates in complex I. The transition between an open and a closed form of complex I is associated with rotation of a helical segment in ND6 and is suggested to represent a step of the catalytic cycle. Asymmetry in the complex III dimer suggests that Q reduced in complex I diffuses preferentially to the nearest binding site in complex III. 45. Brandt U: A two-state stabilization-change mechanism for proton-pumping complex I. Biochim Biophys Acta 2011, 1807:1364-1369. 46. Cabrera-Orefice A, Galemou Yoga E, Wirth C, Siegmund K, Zwicker K, Guerrero-Castillo S, Zickermann V, Hunte C, Brandt U: Locking loop movement in the ubiquinone pocket of complex I disengages the proton pumps. Nat Commun 2018, 9:4500 This study shows, by crosslinking subunit NDUFS7 to the long TMH1-2 loop of ND3, that unrestrestricted movement of the latter loop is required for coupling redox reactions with proton translocation. 47. Guerrero-Castillo S, Baertling F, Kownatzki D, Wessels HJ, Arnold S, Brandt U, Nijtmans L: The assembly pathway of mitochondrial respiratory chain complex I. Cell Metab 2017, 25:128-139. 48. Formosa LE, Dibley MG, Stroud DA, Ryan MT: Building a complex complex: assembly of mitochondrial respiratory chain complex I. Semin Cell Dev Biol 2018, 76:154-162. 49. Lazarou M, McKenzie M, Ohtake A, Thorburn DR, Ryan MT: Analysis of the assembly profiles for mitochondrial- and nuclear-DNA-encoded subunits into complex I. Mol Cell Biol 2007, 27:4228-4237.
35. Verkhovsky M, Bloch DA, Verkhovskaya M: Tightly-bound ubiquinone in the Escherichia coli respiratory complex I. Biochim Biophys Acta 2012, 1817:1550-1556.
50. Kmita K, Wirth C, Warnau J, Guerrero-Castillo S, Hunte C, Hummer G, Kaila VR, Zwicker K, Brandt U, Zickermann V: Accessory NUMM (NDUFS6) subunit harbors a Zn-binding site and is essential for biogenesis of mitochondrial complex I. Proc Natl Acad Sci U S A 2015, 112:5685-5690.
36. Wikstro¨m M, Sharma V, Kaila VR, Hosler JP, Hummer G: New perspectives on proton pumping in cellular respiration. Chem Rev 2015, 115:2196-2221.
51. Scha¨gger H, Pfeiffer K: Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 2000, 19:1777-1783.
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52. Vonck J, Scha¨fer E: Supramolecular organization of protein complexes in the mitochondrial inner membrane. Biochim Biophys Acta 2009, 1793:117-124.
62. Senkler J, Senkler M, Braun HP: Structure and function of complex I in animals and plants - a comparative view. Physiol Plant 2017, 161:6-15.
53. Sousa JS, Mills DJ, Vonck J, Ku¨hlbrandt W: Functional asymmetry and electron flow in the bovine respirasome. eLife 2016, 5:e21290.
63. Schuller JM, Birrell JA, Tanaka H, Konuma T, Wulfhorst H, Cox N, Schuller SK, Thiemann J, Lubitz W, Setif P et al.: Structural adaptations of photosynthetic complex I enable ferredoxindependent electron transfer. Science 2019, 363:257-260 Cryo-EM structure of a cyanobacterial NDH found in photosynthetic organisms including fast kinetics showing that ferredoxin, and not NAD(P)H, is the electron donor for NDH and participates in cyclic electron flow.
54. Davies KM, Blum TB, Ku¨hlbrandt W: Conserved in situ arrangement of complex I and III2 in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants. Proc Natl Acad Sci U S A 2018, 115:3024-3029 Cryo-electron tomography of mitochondrial inner membrane showing conserved respiratory supercomplex organisation for complex I and III, but not complex IV, between mammals, plants and fungi. 55. Blaza JN, Serreli R, Jones AJ, Mohammed K, Hirst J: Kinetic evidence against partitioning of the ubiquinone pool and the catalytic relevance of respiratory-chain supercomplexes. Proc Natl Acad Sci U S A 2014, 111:15735-15740. 56. Fedor JG, Hirst J: Mitochondrial supercomplexes do not enhance catalysis by quinone channeling. Cell Metab 2018, 28:525-531.e4. 57. Milenkovic D, Blaza JN, Larsson NG, Hirst J: The enigma of the respiratory chain supercomplex. Cell Metab 2017, 25:765-776. 58. Hirst J: Open questions: respiratory chain supercomplexeswhy are they there and what do they do? BMC Biol 2018, 16:111. 59. Brandt U: Adaptations of an ancient modular machine. Science 2019, 363:230-231 Concise overview of the complex I superfamily. 60. Schut GJ, Zadvornyy O, Wu CH, Peters JW, Boyd ES, Adams MW: The role of geochemistry and energetics in the evolution of modern respiratory complexes from a proton-reducing ancestor. Biochim Biophys Acta 2016, 1857:958-970. 61. Battchikova N, Eisenhut M, Aro EM: Cyanobacterial NDH-1 complexes: novel insights and remaining puzzles. Biochim Biophys Acta 2011, 1807:935-944.
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64. Laughlin TG, Bayne AN, Trempe JF, Savage DF, Davies KM: Structure of the complex I-like molecule NDH of oxygenic photosynthesis. Nature 2019, 566:411-414 Cyanobacterial NDH cryo-EM structure suggesting that there could be two ferredoxin binding sites in order to deliver electrons to the complex. The structure also describes an intriguing large density which could be an unknown metal cofactor situated close to the plastoquinone binding site. 65. Yu H, Wu CH, Schut GJ, Haja DK, Zhao G, Peters JW, Adams MWW, Li H: Structure of an ancient respiratory system. Cell 2018, 173:1636-1649 Structure of a hydrogen gas-evolving membrane bound hydrogenase (MBH) from the hyperthermophilic archaeon Pyrococcus furiosus highlighting the evolutionary relationship between MBH, Mrp Na+/H+ antiporters and complex I. 66. Roessler MM, King MS, Robinson AJ, Armstrong FA, Harmer J, Hirst J: Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance. Proc Natl Acad Sci U S A 2010, 107:1930-1935. 67. Angerer H, Scho¨nborn S, Gorka J, Bahr U, Karas M, Wittig I, Heidler J, Hoffmann J, Morgner N, Zickermann V: Acyl modification and binding of mitochondrial ACP to multiprotein complexes. Biochim Biophys Acta 2017, 1864:1913-1920. 68. Chovancova E, Pavelka A, Benes P, Strnad O, Brezovsky J, Kozlikova B, Gora A, Sustr V, Klvana M, Medek P et al.: CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol 2012, 8:e1002708.
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ScienceDirect Respiratory complex I — structure, mechanism and evolution Kristian Parey1,2,3,5, Christophe Wirth4,5, Janet Vonck2 and Volker Zickermann1,3 Respiratory complex I is an intricate multi-subunit membrane protein with a central function in aerobic energy metabolism. During the last years, structures of mitochondrial complex I and respiratory supercomplexes were determined by cryo-EM at increasing resolution. Structural and computational studies have shed light on the dynamics of proton translocation pathways, the interaction of complex I with lipids and the unusual access pathway of ubiquinone to the active site. Recent advances in understanding complex I function include characterization of specific conformational changes that are critical for proton pumping. Cryo-EM structures of the NADH dehydrogenase-like (NDH) complex of photosynthesis and a bacterial membrane bound hydrogenase (MBH) have provided a broader perspective on the complex I superfamily. Addresses 1 Institute of Biochemistry II, University Hospital, Goethe University, Frankfurt am Main, Germany 2
Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany 3 Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe University, Frankfurt am Main, Germany 4 Institute of Biochemistry and Molecular Biology, ZBMZ, Medical Faculty, Albert-Ludwigs-University Freiburg, Freiburg, Germany Corresponding authors: Vonck, Janet ([email protected]), Zickermann, Volker ([email protected]) 5 Contributed equally. Current Opinion in Structural Biology 2020, 63:1–9 This review comes from a themed issue on Membranes
the inner membrane of mitochondria or the cell membrane of bacteria [1,2]. The reaction is tightly coupled and reversible. Complex I dysfunction is associated with numerous neuromuscular or neurodegenerative diseases in humans [3,4]. Under conditions that promote reverse electron flow, complex I is a major source of deleterious reactive oxygen species (ROS) [5]. The reversible active (A) to deactive (D) transition [6] is thought to protect against excessive ROS formation [7]. Complex I is one of the largest membrane protein complexes. It is L-shaped with a peripheral arm involved in electron transfer from NADH to Q and a membrane arm responsible for proton pumping (Figure 1). It consists of 14 central subunits, 7 in the peripheral arm and 7 in the membrane arm, that are conserved from bacteria to humans, and about 30 accessory subunits that occur almost exclusively in eukaryotic complex I. We will here use the subunit designations for human complex I throughout. The structures of respiratory complex I from bacteria and from the aerobic yeast Yarrowia lipolytica were solved by X-ray crystallography [8–10]. Since the resolution revolution in cryo-EM [11], structures of mammalian and Y. lipolytica complex I and respiratory supercomplexes were determined by this technique at increasing resolution [12–15,16,17,18,19,20,21]. High-resolution structures have enabled molecular modeling and molecular dynamics simulation approaches that will be indispensable for unravelling the mechanism of redox-linked proton translocation [22,23].
Edited by Beili Wu and Fei Sun
https://doi.org/10.1016/j.sbi.2020.01.004
In this review we focus on progress in structure determination, functional studies and molecular modelling of complex I, its supercomplexes and related structures over the past three years.
0959-440X/ã 2020 Elsevier Ltd. All rights reserved.
Substrate binding sites
Introduction In aerobic energy metabolism, ATP is largely provided by oxidative phosphorylation. The redox-driven proton translocating complexes of the respiratory chain generate the proton motive force that drives ATP synthase. Respiratory complex I (proton pumping NADH:ubiquinone oxidoreductase) couples electron transfer from NADH to ubiquinone (Q) with translocation of four protons across www.sciencedirect.com
The initial electron acceptor in complex I is a flavin mononucleotide (FMN) in subunit NDUFV1 (Figure 1). Binding of substrate close to FMN was shown by X-ray crystallography and cryo-EM [20,24]. A chain of seven FeS clusters connects the NADH binding site with the Q reduction site (Figure 1) [25]. Cluster N1a in NDUFV2 is thought to have a special function because it is located on the distal side of FMN (Figure 1). Based on a series of high-resolution structures of a NDUFV1/2 fragment, a recent study suggests that the redox-state of N1a controls access of NADH and thus protects against overreduction of complex I [26]. Current Opinion in Structural Biology 2020, 63:1–9
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Figure 1
The topology of the Q reduction site in complex I is remarkably different from that of Q reactive sites in other membrane protein complexes. FeS cluster N2, the immediate reductant for Q, is buried in the peripheral arm above the membrane surface (Figure 1) [27,28]. Hydrophobic Q has to move in a tunnel from the lipid bilayer to the Q reduction site (Figure 2a) [9,10]. Two recent cryoEM maps showed that in the region around the tunnel entrance, subunits of the hydrophilic peripheral arm bind lipid head groups, and lipid tails wrap around the hydrophobic surfaces of amphipathic helices lining the membrane/peripheral arm interface region (Figure 2b) [18,21]. This unusual lipid-protein arrangement is thought to cause thinning of the lipid bilayer to facilitate entry of Q into the tunnel and flipping of the Q head group for fast alternating access to Q reactive sites in complexes I and III (Figure 3b) [21]. In the active site, the Q head group is bound in electron transfer distance to cluster N2 by a tyrosine and a histidine residue (Figure 2a) [9,29]. The long isoprenoid tail is thought to be important for guiding and facilitating movement of Q in the extended access pathway [30]. Computational approaches identified multiple intermediary binding sites in the Q tunnel (Figure 2a) [31,32,33]. In [31], site 1 corresponds to the binding position in the active site described above. Site 1matches the binding site for a Q antagonistic inhibitor identified by X-ray crystallography [10]. Site 2 is close to a bend of the tunnel that is surrounded by a remarkable network of charged residues. This network represents the origin of the so-called hydrophilic axis [9] extending towards the proton pumps. Sitedirected mutagenesis and simulations identified critical residues in a loop of NDUFS7 lining the Q access pathway and the interface of the Q and P modules [34]. Recently, cryo-EM showed binding of a native Q molecule to site 2 in Y. lipolytica complex I [21] that might represent a substrate molecule on its way between
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Q-module N-side matrix P-side IMS P-module Current Opinion in Structural Biology
Overall structure and schematic representation of respiratory complex I. (a) Cryo-EM density map of Y. lipolytica complex I (EMD-4873) with the densities of the distinct subunits in different colors. (b) Cryo-EM structure of Y. lipolytica complex I (PDB ID: 6RFR). Central subunits are labelled, the position of FMN (NADH oxidation site) and of FeS clusters Current Opinion in Structural Biology 2020, 63:1–9
N2 (Q reduction site) are highlighted; FMN and FeS clusters are shown in the inset (assignment of EPR signatures N1 to N5 according to Roessler et al. [66]); a native Q molecule is bound in the access pathway [21]. Accessory subunits are shown transparent. Further notable cofactors are represented: the phosphopantetheine cofactor of acyl carrier proteins with appended fatty acid (PP) [67], the NADPH molecule in NDUFA9 and the zinc ion (Zn) in NDUFS6. (c) The central subunits are assigned to functional modules for NADH oxidation (N module), ubiquinone reduction (Q module) and proton translocation (P module). The N and Q modules form the so-called matrix or peripheral arm, the P module corresponds to the membrane arm. Electrons are transferred from FMN to the Q reduction site by a chain of seven FeS clusters. The three antiporter-like subunits ND2 (purple), ND4 (cyan), and ND5 (deep blue) harbor protontranslocation pathways each consisting of two half-channels connecting to the N-side (mitochondrial matrix) and the P-side (intermembrane space, IMS), respectively; the position of a putative fourth proton channel is discussed. Each antiporter-like subunit contains two discontinuous helices (TMH 7a/b, TMH 12a/b), a central lysine residue in the center of the H+ translocation pathway, and a Lys-Glu pair between helices 7 and 5 implicated in lateral signal transfer. The protonation state of critical residues is thought to control the dynamic hydration and thus connectivity of half channels [23,38,39]. Switching between a p-bulge and a regular a-helix was observed for TMH3 of ND6 (orange) [18,44]. www.sciencedirect.com
Structure and function of respiratory complex I Parey et al.
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Figure 2
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Current Opinion in Structural Biology
Q tunnel and lipids. (a) The Q tunnel (gray) in complex I from Y. lipolytica (PDB ID: 6RFR) was calculated using CAVER [68]. Free energy calculations and simulations identified multiple intermediate binding sites in the Q tunnel [31,32,33]; structural evidence for binding of Q to positions matching sites 1 [9], 10 [10,20], and 2 [21] was provided by X-ray crystallography and cryo-EM. The Q head group in site 1 was fitted manually according to Figure 4 of Ref. [9]. (b) A total of 33 phospholipids were modelled in the density map of complex I from Y. lipolytica [21]. For orientation, the Q molecule in site 2 is shown in blue. Lipid head groups in the interface region are shifted towards a plateau above the membrane surface level defined by lipid head groups in the rest of the membrane arm; the lower panel shows a view from the backside of complex I. In the interface region lipid tails are bent and wrapped around the hydrophobic surface of amphipathic helices [21]. Figure 3
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The respirasome (supercomplex I1III2IV1). (a) Cryo-EM map of the respirasome from sheep (EMD-8130) [14]; side view (upper) and view from the matrix (lower), complex I (orange), dimer of complex III (red), complex IV (green). In the lower panel, accessory complex I subunit NDUFA11 providing contact in the membrane plane is highlighted in magenta. (b) Schematic representation of the respirasome with cofactors highlighting the electron (solid arrows) and substrate transfer (dotted arrows) in the supercomplex; asymmetry observed in Q binding to complex III suggests that only the Qo site proximal to complex I is active [44]. Electrons can, however, also be transferred to the Qi site of the other monomer (light blue arrows). Mobility of the Rieske FeS protein (ISP) is critical for bifurcation of electrons at the Qo site; in a bovine respirasome, density indicating rigid protein structure was visible for the distal but not for the proximal ISP, suggesting that the proximal site is active while the distal site is in resting state [53]. www.sciencedirect.com
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the membrane and the active site (Figure 2a). Alternatively, a bound Q might act as a mobile electron carrier shuttling electrons from the Q reduction site to the membrane Q pool [35]. The mechanistic implications of a shuttling Q and electron transfer at or close to site 2 were discussed [32,36]. However, the functional significance of the observed Q binding site remains unresolved and has to be evaluated in future work.
Dynamic water chains and long-range energy transfer in the membrane arm In complex I, the reduction of Q triggers the translocation of protons across the membrane, remote from the Q binding site. The three largest membrane arm subunits ND2, ND4 and ND5 have a common core fold [8] and are related to subunits of Mrp-type sodium proton antiporters [37]. In each subunit, TMH7 and TMH12 are discontinuous (Figure 1c) [8]. Proton-translocation pathways extend along TMH segments 7b and 12b and connect the N and the P side with the central membrane plane [8–10] (Figure 1c). The position of a fourth proton translocation pathway is debated. Recent computational work suggests that channel hydration is dynamic and dependent on the charge state of key residues positioned at the origin of each half channel (Figure 1c) [38,39,40]. These residues line up along the hydrophilic axis that forms a continuous connection of protonatable residues to the Q reduction module (Figure 1c). Q reduction is thought to drive initial proton uptake in ND1 [41], which in turn triggers a signal along the hydrophilic axis towards ND5 [23]. The signal is conveyed by a series of lateral proton transfers and switching of a LysGlu ion pair in each antiporter-like subunit. It controls the concerted formation and decay of water wires and drives vectorial proton movements while back leak reactions are prevented [23]. The described mechanism is still hypothetical but recent structural work identified conformational alterations in TMH8 of the antiporter-like subunits that might reflect changes linked with signal propagation in the hydrophilic axis [18].
form shows a localized unfolding of protein structure around the Q reduction site [12,18,19]. Restructuring during transition into the A form is thought to occur by an induced fit mechanism with Q acting as a template [19]. In the membrane arm, a p-bulge in TMH3 of ND6 is exclusively present in the D form [18]. The interconversion into an extended helix present in the A form is associated with a rotation of the C-terminal helix segment, which becomes visible as a prominent change of side chain positions of two aromatic residues [18]. Interestingly, the p-bulge of TMH3 of ND6 is connected to charged residues of the E channel and is therefore an element of a putative proton translocation pathway (Figure 1c) [18]. The extensive conformational rearrangements in the Q site and in ND6 were not observed in complex I from Y. lipolytica [20]. This is consistent with a lower energy barrier for the transition between A and D form in the yeast enzyme complex [42]. The absence of extensive unfolding around the Q site might be explained by the presence of a Q molecule in site 2 [21], which was not observed in other complex I structures so far.
Structures of complex I in different functional states
A recent study of ovine I1III2 supercomplex showed the presence of two major complex I classes called open and closed state in one data set [44]. The closed state largely matches the A form of bovine and mouse complex I (see above); the open state represented an ensemble of states and showed extensive similarity with the D form. However, biochemical analysis indicated the absence of detectable amounts of the D form in the ovine supercomplex preparation. Based on this finding the authors of this study concluded that the conversion between open and closed state occurs while complex I is in the A form and is therefore part of the catalytic cycle [44]. They suggest that the remarkable rotation of the TMH3 segment of ND6 is an efficient element of conformational coupling within the E pathway [44]. The D form is proposed to represent one of the open-like states with more extensive unfolding of loops around the Q site [44]. Although this study provides exciting new insights, it has to be taken with a grain of salt as the data for evaluating the relative amounts of A and D form in the preparation were not shown.
Complex I from vertebrates and fungi exists either in the A or D form [7,42]. The A form is able to catalyze fast electron transfer to Q. When substrates sustaining complex I turnover are depleted, e.g. during ischemia, complex I spontaneously switches to the resting D form. Return to the A form occurs by slow activating turnovers. This process is impeded or blocked by divalent cations, alkaline pH or covalent modification of a specific cysteine residue in subunit ND3 [43]. Modulation of the A/D transition by thiol reactive agents offers new strategies for mitigating tissue damage in myocardial infarction [5]. The structural basis of the A/D transition was investigated by cryo-EM in bovine and mouse complex I [12,18,19]. The A and D form are related by slight opposing rotations of the matrix arm and a large sector of the membrane arm [12]. The D
The structure of complex I from Y. lipolytica was analyzed by cryo-EM in the deactive state and after freezing the enzyme under turnover [20]. Several lines of evidence indicate that electron transfer from N2 to Q occurs while the Q head group is bound to a tyrosine and a histidine residue close to cluster N2 [9,29]. However, in the cryoEM structure under turnover, a Q molecule was modelled in a different position between the tip of the b1b2 loop and helix 2 of NDUFS7 [20]. The Q position observed overlaps with the binding site of an inhibitor determined by X-ray crystallography [10] and matches one of the sites identified in molecular simulations (site 1) (Figure 2a) [31]. These data support a two-state stabilization change mechanism [45] that postulates cycling between two Q
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Structure and function of respiratory complex I Parey et al.
binding positions permitting either electron transfer (E state) or protonation steps (P state) in the reduction of Q. The rearrangement of the Q binding site associated with stabilization of negatively charged Q intermediates is thought to generate the power stroke that is transmitted to the membrane domain to drive the proton pumps [20,45]. The structural data also concur with free energy calculations predicting that after formation in site 1, QH2 relaxes to site 1[31].
Identification of mobile loop elements at the membrane/matrix arm interface We have proposed that the redox chemistry of Q drives the concerted rearrangement of three loops in NDUFS2, ND3 and ND1 located in and around the Q reduction site [10,20]. The NDUFS2 loop harbors the conserved histidine that binds the Q head group in the active site. Modelling in different conformations in a variety of structures indicates flexibility of the loop. The nearby TMH5-6 loop of ND1 carries several conserved acidic residues and represents the endpoint of the hydrophilic axis connecting to the proton pumps. The ND3 loop carries a cysteine residue that is employed as a reporter group for the A/D transition because it shows selective accessibility, for example, for NEM labeling, in the D form only [43]. Introducing a cysteine residue in helix 2 of NDUFS7 by site-directed mutagenesis permitted formation of a disulfide bond with the ND3 cysteine [46]. Electron transfer was essentially unaffected but proton pumping stopped after formation of the disulfide bond, indicating that unrestricted movement of the ND3 loop is essential for the coupling mechanism [46].
Structure of a complex I assembly intermediate Complex I assembly is a convoluted process that is supported by assembly factors [47,48]. Assembly factor NDUFAF2 has been associated with attachment of the N module to nascent complex I [49]. Deletion of the gene for accessory subunit NDUFS6 in Y. lipolytica stalled complex I assembly and blocked release of NDUFAF2 [50]. The cryo-EM structure of the assembly intermediate was recently determined and offered clues on the interplay of NDUFAF2 with accessory subunits NDUFS4, NDUFS6 and NDUFA12 in the last step of complex I assembly [21].
Supercomplexes Respiratory supercomplexes were initially isolated by blue-native PAGE after mild solubilization of mitochondrial membranes by digitonin [51], and later visualized by single-particle electron microscopy [52]. In the last few years cryo-EM structures have been determined from several mammalian species (Figure 3a) [14,15,16,17,44,53]. Most of these structures have the composition I1III2IV1, the so-called respirasome, that together with Q and cyt c contains all components www.sciencedirect.com
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to transfer electrons from NADH to oxygen (Figure 3b). The basic architecture of the respirasome is the same in all species, with complex III nestled at the concave side of the complex I membrane arm and complex IV attached at the distal end. Intercomplex contacts are established by accessory subunits, most notably NDUFA11 between complex I and III (Figure 3a). The supercomplex structures are usually not very rigid and in some studies multiple conformations are found [14,53]. A cryo-electron tomography study [54] showed that the in vivo arrangement of complexes I and III is conserved not only in mammals, but also in yeast (Y. lipolytica) and plant mitochondria, but that the position of complex IV is variable between and within species. The supercomplex structures show that there is no intraprotein path for Q between complexes I and III and experimental work supports the availability of reduced Q outside the supercomplex [55,56]. These data provide strong evidence against substrate channelling. However, one of the Q binding sites of complex III faces complex I, leading to a short diffusion pathway, and two cryo-EM studies have found evidence for asymmetry in complex III that supports preferential Q entry at this site (Figure 3) [44,53]. There is no evidence that supercomplex formation would play a role in ROS production [57]. A recent hypothesis states that supercomplexes have evolved to ensure a homogeneous distribution of complexes in the crowded environment of the mitochondrial inner membrane to promote an efficient and rapid catalysis of respiration [58].
Complex I superfamily Complex I is a member of a superfamily of membranebound redox enzymes that have adapted ancient modules to cope with diverse evolutionary pressures [59,60]. Recently, structures of some of these complexes have been determined by cryo-EM, providing a structural basis for the evolutionary relationships in this family. Cyanobacteria are the organisms that first evolved oxygenic photosynthesis. They contain several enzymes from the complex I superfamily with diverse functions, including respiration, cyclic electron flow and CO2 acquisition [61]. The cyanobacterial NDH-1L complex is the evolutionary origin of the NADH dehydrogenase-like (NDH) complex in chloroplasts. This ‘photosynthetic complex I’ should not be confused with respiratory complex I of plant mitochondria [62]. Two cryo-EM studies of NDH1L from Thermosynechococcus elongatus [63,64] now shed light on its structure (Figure 4b). The NDH complex is involved in cyclic electron flow, transferring electrons from ferredoxin, reduced by photosystem-1, to plastoquinone and pumping protons across the thylakoid membrane, thereby adjusting the ATP/NADPH ratio produced by photosynthesis. It shares 11 of the 14 core subunits of respiratory complex I, only lacking the three subunits of the N module that transfer electrons from Current Opinion in Structural Biology 2020, 63:1–9
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Figure 4
(a)
(b)
(c)
(d)
Current Opinion in Structural Biology
Conserved structural and functional elements in complex I and related complexes. Structures and schematic representations of (a) MBH complex from P. furiosus (PDB ID: 6CFW, EMD-7468) (b) NDH complex from T. elongatus (PDB ID: 6HUM EMD-0281) (c) bacterial complex I from T. thermophilus (PDB ID: 4HEA) and (d) mitochondrial complex I from mouse (PDB ID: 6G2J, EMD-4345); coloring of central subunits reflects evolutionary relations between the different subunits of the complexes (note that MbhI is related to ND3 and ND5). Accessory subunits are shown in gray. The loop cluster at the interface of Q and P module in complex I is conserved in NDH and MBH. Movement of the ND3 loop (yellow) was shown to be essential for coupling Q reduction to proton translocation [46].
NADH to the Q binding site (Figure 4b). Instead, four oxygenic-photosynthesis-specific subunits facilitate electron transfer from ferredoxin to plastoquinone. Some anaerobic archaea have a hydrogen gas-evolving membrane-bound hydrogenase (MBH) that uses ferredoxin as electron donor to reduce protons to molecular hydrogen [60]. MBH is a sodium pump, generating a Current Opinion in Structural Biology 2020, 63:1–9
sodium gradient that is used to energise a sodium-driven ATP synthase. The cryo-EM structure of the 14-subunit, 300 kDa MBH from Pyrococcus furiosus [65] (Figure 4a) shows a proton-translocating unit similar to the ND2/ ND6/ND4L unit of complex I (Figure 4c). A putative four-subunit sodium-translocating module with homology to Mrp H+/Na+ antiporter subunits is located at the distal end of the membrane domain. This topology www.sciencedirect.com
Structure and function of respiratory complex I Parey et al.
suggests that the redox reaction is initially coupled to generation of a proton gradient, which in turn drives Na+ translocation [65]. Interestingly, the membrane anchored hydrogenase module, consisting of four orthologs of the complex I Q module and an ortholog of ND1, is attached to the opposite end of the proton-pumping unit compared to complex I (Figure 4a,c,d). MBH has a functional, H2 evolving [NiFe] hydrogenase unit, MbhL and MbhJ with a NiFe center and an FeS cluster, respectively, while the related subunits (NDUFS2 and NDUFS7) in complex I have lost the NiFe center and are involved in Q reduction. This supports the proposed evolution of complex I from an H2-evolving ancestor [60]. Intriguingly, loops critical for coupling of Q reduction to proton pumping already have equivalents in MBH, suggesting a conservation of the coupling mechanism despite extensive differences in the redox-chemistry.
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Fiedorczuk K, Sazanov LA: Mammalian mitochondrial complex I structure and disease-causing mutations. Trends Cell Biol 2018, 28:835-867.
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Chouchani ET, Methner C, Nadtochiy SM, Logan A, Pell VR, Ding S, James AM, Cocheme HM, Reinhold J, Lilley KS et al.: Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I. Nat Med 2013, 19:753-759.
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Kotlyar AB, Vinogradov AD: Slow active/inactive transition of the mitochondrial NADH-ubiquinone reductase. Biochim Biophys Acta 1990, 1019:151-158.
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Dro¨se S, Stepanova A, Galkin A: Ischemic A/D transition of mitochondrial complex I and its role in ROS generation. Biochim Biophys Acta 2016, 1857:946-957.
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Efremov RG, Sazanov LA: Structure of the membrane domain of respiratory complex I. Nature 2011, 476:414-420.
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Baradaran R, Berrisford JM, Minhas GS, Sazanov LA: Crystal structure of the entire respiratory complex I. Nature 2013, 494:443-448.
Conclusions
11. Ku¨hlbrandt W: The resolution revolution. Science 2014, 343:1443-1444.
The revolution in cryo-EM technology triggered tremendous progress in understanding the structure of mitochondrial complex I and respiratory supercomplexes. Structural characterization of complex I in different functional states is now an area of intense research and molecular simulation approaches and functional studies have provided exciting new insights into the inner workings of this intricate molecular machine. Common structural features in evolutionary related enzyme complexes offer clues on a universal coupling mechanism but also identify specific adaptations within the complex I superfamily. Despite this amazing gain of knowledge, consensus on the molecular mechanism of redox-linked proton translocation is still lacking. The combination of different experimental approaches is needed to obtain a consistent and comprehensive picture in the future.
Conflict of interest statement Nothing declared.
Acknowledgements This work was supported by the German Research Foundation (ZI 552/4-2 and ZI 552/5-1 to V.Z.) and by the Max Planck Society.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Zickermann V, Wirth C, Nasiri H, Siegmund K, Schwalbe H, Hunte C, Brandt U: Mechanistic insight from the crystal structure of mitochondrial complex I. Science 2015, 347:44-49.
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structure of an assembly intermediate provide molecular insights into the cause of complex I-linked Leigh syndrome and into complex I assembly. 22. Haapanen O, Sharma V: A modeling and simulation perspective on the mechanism and function of respiratory complex I. Biochim Biophys Acta 2018, 1859:510-523. 23. Kaila VRI: Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I. J R Soc Interface 2018, 15:20170916 This review article presents a detailed mechanistic model for redox-linked proton translocation in complex I. 24. Berrisford JM, Sazanov LA: Structural basis for the mechanism of respiratory complex I. J Biol Chem 2009, 284:29773-29783. 25. Sazanov LA, Hinchliffe P: Structure of the hydrophilic domain of respiratory complex I from Thermus thermophilus. Science 2006, 311:1430-1436. 26. Schulte M, Frick K, Gnandt E, Jurkovic S, Burschel S, Labatzke R, Aierstock K, Fiegen D, Wohlwend D, Gerhardt S et al.: A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I. Nat Commun 2019, 10:2551 This study describes several high resolution structures of the electron input domain and provides evidence for a molecular switch that prevents overreduction of complex I. 27. Efremov RG, Baradaran R, Sazanov LA: The architecture of respiratory complex I. Nature 2010, 465:441-445. 28. Hunte C, Zickermann V, Brandt U: Functional modules and structural basis of conformational coupling in mitochondrial complex I. Science 2010, 329:448-451. 29. Tocilescu MA, Fendel U, Zwicker K, Dro¨se S, Kerscher S, Brandt U: The role of a conserved tyrosine in the 49-kDa subunit of complex I for ubiquinone binding and reduction. Biochim Biophys Acta 2010, 1797:625-632. 30. Fedor JG, Jones AJY, Di Luca A, Kaila VRI, Hirst J: Correlating kinetic and structural data on ubiquinone binding and reduction by respiratory complex I. Proc Natl Acad Sci U S A 2017, 114:12737-12742 Study investigating the kinetics of complex I with Q ranging from Q1 to Q10 and offering clues on the Q access pathway and the importance of the long isoprenoid chain of the physiological substrate. 31. Warnau J, Sharma V, Gamiz-Hernandez AP, Di Luca A, Haapanen O, Vattulainen I, Wikstro¨m M, Hummer G, Kaila VRI: Redox-coupled quinone dynamics in the respiratory complex I. Proc Natl Acad Sci U S A 2018, 115:E8413-E8420 This in silico study identifies intermediate Q binding sites within the extended Q cavity and shows that the dynamics of Q is redox-state dependent. 32. Haapanen O, Djurabekova A, Sharma V: Role of second quinone binding site in proton pumping by respiratory complex I. Front Chem 2019, 7:221 http://dx.doi.org/10.3389/fchem.2019.00221 Molecular dynamics simulations suggesting that the second Q binding site, remote from cluster N2, could play a key role in the catalytic cycle. 33. Teixeira MH, Arantes GM: Balanced internal hydration discriminates substrate binding to respiratory complex I. Biochim Biophys Acta 2019, 1860:541-548 This study uses computational approaches to understand the impact of hydration of the unusual quinone binding cavity in complex I and isoprenoid chain length of quinone. 34. Galemou Yoga E, Haapanen O, Wittig I, Siegmund K, Sharma V, Zickermann V: Mutations in a conserved loop in the PSST subunit of respiratory complex I affect ubiquinone binding and dynamics. Biochim Biophys Acta 2019, 1860:573-581 This study combines site-directed mutagenesis and computational approaches to assess the function of a loop in NDUFS7 for access of Q and coupling of Q reduction to proton pumping.
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