Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia–reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the ‘active’ state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the ‘deactive’ state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I.
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We thank the staff at the Astbury Biostructure Laboratory, University of Leeds (funded by the University of Leeds and Wellcome Trust 108466/Z/15/Z) and at the UK National Electron Bio-Imaging Centre (eBIC) at the Diamond Light Source for assistance with cryo-EM data collection, the staff at the Phenomics Animal Care Facility, S. Ding and I. Fearnley (Cambridge) for mass spectrometry analyses, and M. Hartley and A. Raine (Cambridge) for IT support. This work was supported by Medical Research Council grant numbers MC_U105663141 (J.H.), MC_UU_00015/2 (J.H.) and MC_UU_00015/5 (C.V.).
Integrated supplementary information
Supplementary Figure 1 Resolution estimates of the maps for the active and deactive states of complex I.
a, b: data on the active complex. c, d: data on the deactive complex. The estimated resolutions, defined where the line at FSC = 0.143 crosses the red curve, are 3.3 Å for the active complex (a) and 3.9 Å for the deactive complex (c). In both cases the refined models agree well with the maps, as shown by the map vs. model FSC curves (blue). In b) and d), local resolutions were estimated using the Local Resolution function in RELION with default parameters and plotted using UCSF Chimera.
Classification and refinement of the cryo-EM density maps for the active and deactive states of mouse complex I.
Example densities (carved at 2 Å radius, threshold level 0.065) drawn from different parts of the map of active mouse complex I.
Example densities (carved at 2 Å radius, threshold level 0.04) of phosholipids from the map of active mouse complex I.
a) Intact mass measurements revealed two masses for NDUFA10, for the unmodified and singly phosphorylated versions (present at the point of measurement). b) Spectrum of fragment ions produced by ETD of a doubly charged precursor ion 942.48 (m/z) with the precursor ion truncated to 10% relative intensity. The observed neutral peptide mass for the phosphorylated peptide is 1882.955 Da, relative to a calculated mass of 1882.959 Da. The observed peptides are mapped onto the amino acid sequence where the phosphorylation site is marked and c is carbamidomethylcysteine. The z7 and z8 fragments confirm Ser36, rather than Ser33, as the site of phosphorylation. No additional phosphorylated peptides from NDUFA10 were observed.
Densities and models for TMH8 in subunits ND2, ND4 and ND5 of mouse complex I (carved at 2 Å radius, threshold level 0.065).
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Nature Communications (2018)