www.nature.com/cr www.cell-research.com ARTICLE Structure of the intact 14-subunit human cytochrome c oxidase Shuai Zong 1, Meng Wu1, Jinke Gu1, Tianya Liu1, Runyu Guo1 and Maojun Yang1,2 Respiration is one of the most basic features of living organisms, and the electron transport chain complexes are probably the most complicated protein system in mitochondria. Complex-IV is the terminal enzyme of the electron transport chain, existing either as randomly scattered complexes or as a component of supercomplexes. NDUFA4 was previously assumed as a subunit of complex-I, but recent biochemical data suggested it may be a subunit of complex-IV. However, no structural evidence supporting this notion was available till now. Here we obtained the 3.3 Å resolution structure of complex-IV derived from the human supercomplex I1III2IV1 and assigned the NDUFA4 subunit into complex-IV. Intriguingly, NDUFA4 lies exactly at the dimeric interface observed in previously reported crystal structures of complex-IV homodimer which would preclude complex- IV dimerization. Combining previous structural and biochemical data shown by us and other groups, we propose that the intact complex-IV is a monomer containing 14 subunits. Cell Research (2018) 28:1026–1034; https://doi.org/10.1038/s41422-018-0071-1 INTRODUCTION states under physiological conditions, either being assembled into Mitochondria are critical to many cellular activities. Respiration is supercomplexes or freely scattered on mitochondrial inner the central function of mitochondria, and is exquisitely regulated membrane.36 NDUFA4 was originally considered as a subunit of in multiple ways in response to varying cell conditions.1–3 The Complex-I37 but was proposed to belong to Complex-IV recently.38 assembly of respiratory chain complexes, Complex I–IV (CI, NADH: However, the precise location of NDUFA4 in CIV remains unknown, ubiquinone oxidoreductase; CII, succinate:ubiquinone oxidoreduc- and thus this notion still lacks structural support. In the present tase; CIII, cytochrome bc1 complex; and CIV, cytochrome c oxidase) study, with the 3.3 Å density map of human CIV, for the first time, into supercomplexes (I1III2IV1–2, III2IV1–2) or even a megacomplex we obtained the entire CIV structure containing 14 subunits, 3–10 (I2III2IV2) is a critical form of respiration regulation. Many lines which includes the extra subunit NDUFA4. In all the previous of evidence show that the formation of higher-order complexes crystal structures, CIV is assembled to form a homodimer with can structurally stabilize the individual complexes,11–15 raise the each protomer containing 13 subunits, while in all supercomplex efficiency of electron transfer,16–22 and significantly reduce the structures CIV exists as a monomer. Here, we found NDUFA4 is generation of reactive oxygen species (ROS).23–25 Functional incorporated into CIV and binds exactly to the dimeric interface in studies in vivo also confirmed the validity of these higher-order the crystal structures of CIV dimer, indicating that NDUFA4 assemblies in living cells. Several recently acquired structures of actually hampered the formation of CIV dimer. Structural analyses supercomplex I1III2IV1 (SCI1III2IV1) and megacomplex I2III2IV2 indicated that the CIV homodimer with each protomer containing provided exciting insights into the function and mechanism of 13 subunits was obtained because of the usage of strong these huge machines and led to several interesting hypoth- detergent cholic acid for crystallization which expelled NDUFA4 eses.4,6,9,10 All these structures, especially that of the megacom- from the supercomplexes. Therefore, CIV should be a 14-subunit plex,10 indicated the existence of Q-pool and the close spatial monomer instead of a homodimer with each protomer containing proximity of the binding sites of cytochrome c in CIII and CIV, 13 subunits which is described by the previous 37 X-ray crystal although no protein subunits were shown to directly form a structures (Supplementary information, Table S1). physical channel that can assist CoQ and cytochrome c transport. CIV is the terminal oxidase of the electron transport chain in mitochondria.26–29 It accepts electrons from cytochrome c to RESULTS AND DISCUSSION reduce the oxygen to water and meanwhile pumps two protons Protein purification and structure determination from the matrix side to the intermembrane space (IMS).30–32 In this study, the human mitochondrial electron transport chain Previous studies suggested that the mammalian CIV consists of 13 supercomplex I1III2IV1 (SCI1III2IV1) was purified from cultured distinct subunits, along with three copper II ions, one magnesium HEK293F cells as previously described.10 Super-resolution images ion, one zinc ion, two heme A molecules, and several phospholipid were collected on a Titan Krios microscope operated at a voltage of molecules.33–35 Current opinions point out that CIV exists in two 300 kV with a K2 direct electron detector instead of the Falcon-II 1Ministry of Education Key Laboratory of Protein Science, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China and 2School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China Correspondence: Maojun Yang ([email protected]) These authors contributed equally: Shuai Zong, Meng Wu, Jinke Gu, Tianya Liu Received: 14 June 2018 Revised: 23 June 2018 Accepted: 3 July 2018 Published online: 20 July 2018 © IBCB, SIBS, CAS 2018 Structure of intact human complex IV S. Zong et al. 1027 1234567890();,: Fig. 1 Overall structure of human CIV monomer. a Cryo-EM density map of human CIV monomer along with the cartoon-presented models after soft mask subregion refinement being aligned to the 3.3 Å map are shown in two differently rotated views along the membrane. b The overall structure of human CIV. Subunits are colored and labeled with text in the same colors, respectively. The cofactors and phospholipids are shown in spheres. CDL, cardiolipin; PEE, phosphatidylethanolamine. The transmembrane region is indicated by two dashed lines. M, matrix; IM, inner membrane; IMS, intermembrane space detector that we used before.10 The process of 3D reconstruction might belong to CIV.38 In all previous crystal structures, CIV alone was described in Supplementary information, Figure S1. Further forms a homodimer, however, in all supercomplex structures, CIV refinement with the mask encircling CIV results in a 3.3 Å resolution exists as a monomer.4–6,9 In the present study, we got the 3.3 Å map of CIV (Supplementary information, Figures S1 and S2). Using a density map of CIV using cryo-EM single particle reconstruction combination of structure docking and de novo modeling, we method to unequivocally assign NDUFA4 as the 14th subunit of obtained the atomic structure of the CIV. The well-resolved density human CIV (Figs. 1 and 2). Our structural and biochemical data maps allowed us to build the structure models of almost all the confirmed that the CIV either from a supercomplex or at a residues with side chains in COOT39 (Fig. 1; Supplementary monomer state contains the NDUFA4 subunit (Fig. 3). Thus, we information, Figure S3 and Tables S2 and S3). The “gold standard” have determined the first structure of the intact human CIV Fourier shell correlation (FSC) criteria between the models and maps containing 14 distinct subunits. have no significant differences, indicating the absence of overfitting The 13 traditional subunits of human CIV adopt similar (Supplementary information, Figure S3g). In the final CIV structure, arrangements as in other mammalian CIV structures reported we could accurately place 14 subunits, including NDUFA4, as well as previously.33 Intriguingly, NDUFA4 is identified in our final a multitude of cofactors, including 4 phospholipids, 2 heme structure. The N- and C-terminals of the NDUFA4 are located at molecules,2coppercenters,and1magnesiumion(Fig.1b). Among the matrix side and the IMS, respectively. The middle region of 1888 residues of the mature human CIV, 1,857 (98.36%) residues NDUFA4 forms a transmembrane helix (TMH) that parallels the were modeled in our structure and 1824 (96.61%) residues were main TMHs of MT-CO1 (mitochondrially encoded cytochrome c assigned with side chains (Supplementary information, Figure S2). oxidase I) and is adjacent to the TMH2 of MT-CO2 (Figs. 1b and 2). The head of a well-defined CDL (cardiolipin) molecule inserts into Overall structure of 14-subunit human CIV the deep pocket formed by the main TMHs of MT-CO1, MT-CO3, Although numerous structures of CIV from different species have the C-terminal region and the TMH of NDUFA4, and Cox6B1 at the been determined in the past two decades, none identified the IMS side (Figs. 1b and 4). This CDL molecule might be important position of the NDUFA4 subunit (Supplementary information, for stabilizing NDUFA4 in CIV, as this position is occupied by a Table S1). NDUFA4 was originally considered as an accessory cholic acid and a phosphatidylglycerol (PGV) molecule in most subunit of CI,37 but further studies indicated that this protein crystal structures of the CIV dimer (Fig. 4b). By contrast, the other Cell Research (2018) 28:1026 – 1034 Structure of intact human complex IV S. Zong et al. 1028 Fig. 2 Interactions between NDUFA4 and other subunits of CIV. a Charge-smoothed potential map of CIV generated by PyMOL, except that NDUFA4 is shown in cartoon. Shown in two differently rotated views along the mitochondrial inner membrane. CDL and PEE are shown in stick and colored yellow and blue, respectively. The N- and C-terminals are indicated. b Cryo-EM densities of the NDUFA4 in human CIV monomer, superimposed with atomic model. The N and C terminals are indicated. c Interactions between NDUFA4 and other subunits of CIV. Residues and subunits are labeled and shown in stick and cartoon, respectively. CDL is colored yellow and labeled red.