Structures of Respiratory Supercomplex I+III2 Reveal Functional and Conformational Crosstalk

Abstract

The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III₂ limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII₂. CI shows a transition between "closed" and "open" conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII₂, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII₂, suggesting that interaction with CI disrupts CIII₂ symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.

Keywords: bioenergetics; complex i; cryoEM; cytochrome bc1 complex; mitochondria; oxidative phosphorylation; oxidoreductas; protein structure; respiration; supercomplex.

Graphical abstract

Figure 1

Purification of SC I+III₂ from Ovine Mitochondria (A) Q-Sepharose anion-exchange column chromatogram of LNMG-extracted washed mitochondrial membranes. Chromatograms show A₂₈₀ (blue line), A₄₂₀ (red line), percentage buffer B (containing 1M NaCl) (dashed black line), and CI NADH:FeCy activity (lime green triangles) throughout. Complex (C)I elutes in two distinct peaks: the first peak is isolated CI, and the second peak is SC I+III₂ and excess CIII₂. (B) Superose 6 size exclusion column chromatogram of amphipol-(A8-35)-exchanged SC from the second peak fraction of the anion-exchange step shown in (A) in the absence of detergent. (C) Superose 6 size exclusion column chromatogram of amphipol-stabilized SC I+III₂ from peak fraction containing SC I+III₂ in (B). (D) BN-PAGE gel of the purified SC I+III₂ with isolated CI and CIII₂ shown for comparison. (E) SDS-PAGE of purified SC I+III₂ with labels for some subunits identified by mass spectrometry (MS). Some labels were excluded for clarity. See also Figure S1 and Table S1.

Figure 2

NADH:cyt c Oxidoreductase Activity of Isolated SC I+III₂ (A) Schematic shows positions of CI (blue) and CIII₂ (green) within the SC and the different catalytic sites. The FMN, FeS clusters, and heme groups are shown colored by atom: carbon in gray, nitrogen in blue, oxygen in red, sulfur in yellow, and iron in orange. The two major conformations of the Rieske FeS domain of the CIII subunit UQCRFS1 are indicated with Q_p-proximal in orange and c₁-proximal in red. The gray area indicates the approximate extent of the amphipol-lipid belt. (B) [NADH]-activity curves, NADH oxidation (top), and cyt c reduction (bottom) throughout, in standard buffer (SB) plus 100 μM cyt c. (C) [cyt c]-activity curves in SB plus 100 μM NADH. (D) [DQ]-activity curve in SB plus 100 μM NADH and 100 μM cyt c. (E) [NADH]-activity curves in SB plus 10 μM DQ and 100 μM cyt c added. (F) [cyt c]-activity curves in SB plus 10 μM DQ and 100 μM NADH. (G) [DQ]-activity curves in lipid-detergent (LD) buffer plus 100 μM NADH and 100 μM cyt c. (H) [NADH]-activity curves in LD buffer plus 100 μM DQ and 100 μM cyt c. (I) [cyt c]-activity curves in LD buffer plus 100 μM DQ and 100 μM NADH. Data are mean ± SEM. See also Figure S2 and Table S3.

Figure 3

BSA Uncouples CI and CIII₂ Activity in SC I+III₂, but [DQ] Alone Has Little Effect (A–F) SC I+III₂ activity time courses after addition of 5 μM NADH to SB plus 100 μM cyt c (A); 5 μM NADH to SB plus 100 μM cyt c and 10 μM DQ (B); 10 μM NADH to SB plus 100 μM cyt c and 10 μM DQ (C); 10 μM NADH to SB plus 100 μM cyt c and 100 μM DQ(D); 10 μM NADH to SB plus 100 μM cyt c, 100 μM DQ, and 10 μM BSA (E); and 10 μM NADH to LD buffer plus 100 μM cyt c and 100 μM DQ (F). In each panel, the [NADH] (blue circles) and [reduced cyt c] (red squares) are shown at each time point (n = 8, data are represented as mean ± SEM). Initial rates and final concentrations are denoted by blue lines for [NADH] and red lines for [cyt c] and indicted in the panels (mean ± SEM). Green triangles represent calculated values of DQH₂ at each time point assuming [DQH₂](t) = {[NADH](initial)-[NADH](t)}-{[Red. cyt c](t)/2} and plotted as mean ± SEM. (G) [DQ]-activity curves, NADH oxidation (top), and cyt c reduction (bottom) throughout, in SB plus 10 μM BSA, 100 μM NADH, and 100 μM cyt c. (H) [NADH]-activity curves in SB plus 10 μM BSA, 100 μM DQ, and 100 μM cyt c. (I) [Cyt c]-activity curves in SB plus 10 μM BSA, 100 μM DQ, and 100 μM NADH. (J) [NADH]-activity curves in SB plus 100 μM DQ and 100 μM cyt c. (K) [Cyt c]-activity curves in SB plus 100 μM DQ and 100 μM NADH. (L) NADH oxidation activity in the absence of cyt c at the indicated DQ concentrations in SB: rotenone, 2.5 μM rotenone; BSA, 10 μM BSA; LD buf., lipid-detergent buffer. Data are mean ± SEM, n = 8–12. Not significant (n.s.) indicates p > 0.01. See also Table S3.

Figure 4

SC I+III₂ Structures Reveal State-Dependent Conformational Changes in the CI Membrane Arm (A–D) CryoEM densities for the (A) closed class, (B) open class 1, (C) open class 2, and (D) open class 3. (E) Overlay of the models for the different SC classes aligned by the CI membrane arm shown as cartoons. Models colored as in (A)–(D) and viewed from CI side (left) and the mitochondrial matrix (right). Differences in the relative positions of the CI peripheral arm and CIII₂ are indicated by arrows. (F) Slice through the CI peripheral arm and CIII₂ at the position indicated by the red dashed line in (E). Models are shown as ribbons and colored as in (E). Approximate boundaries between subunits are indicated by black lines. (G) CryoEM density for the closed state CI (left, blue density) and open state 1 (right, green density) for the NDUFS2 β1-β2 loop in the Q-tunnel (top) and the ND3 TMH1-TMH2 loop (bottom). Models are shown as cartoons and colored by subunit: green, NDUFS2; red, NDUFS7; light green, ND1; white, ND3; and pink, ND6. The black and white hexagon indicates approximate binding site for CoQ. (H) ND6 TM3 from the closed state (left, blue) and open state 1 (right, green) viewed from the same side. The π-bulge in the open state 1 is indicated. Models shown as cartoons with side chains as sticks colored by atom, with nitrogen blue, oxygen red, sulfur yellow, and carbon colored as the cartoon helix. (I) View from the mitochondrial matrix looking at ND6 TMH3 in the closed state (top) and open state 1 (bottom). Models shown as cartoons and colored by subunit: ND4L in green, ND6 in light blue, ND3 in blue-green, ND1 in gray, and NDUFS2 in green. Positions of the ND6 TMH3 side chains Tyr70 and Phe68 are shown. See also Figure S4 and Table S4 for initial processing.

Figure 5

Focus-Revert-Classify Strategy Results in at Least Six Different CI Open Structures (A) Schematic of focus-revert-classify strategy for separating CI particles on the basis of the angle between the peripheral and membrane arms. (B) Overlay of CI models generated from the focus-revert-classify strategy aligned by the CI membrane arm. Models colored with the closed state blue and the different open states from dark green to yellow-green according to overall resolution. Viewed from CI side (left) and the mitochondrial matrix (right). Differences in the relative positions of the CI peripheral arm indicated by arrows. (C) Slice through the CI peripheral arm at the position indicated by red dashed line in (B). Models shown as ribbons and colored as in (B). Approximate boundaries between subunits are indicated by black lines, subunits labeled. See also Figure S6.

Figure 6

Interactions, CoQ Density, and Overall Arrangement of the Amphipol-Stabilized SC I+III₂ Particles (A) Interaction between CI and CIII₂ in the membrane. CI subunit NDUFA11 in cyan, CIII₂ subunits UQCRB in green, and UQCRQ in light green. The putative “positive cluster” lipid-binding site is circled and labeled. Side chains of important residues are shown as sticks and colored by atom, with nitrogen blue, oxygen red, and carbon the same color as the subunit. Models shown as cartoons throughout. (B) Interaction between CI and CIII₂ in the mitochondrial matrix. CI subunits NDUFB4 are in cyan and NDUFB9 in light blue, and CIII₂ subunit UQCRC1 are in light green. Important side chains are shown and colored as in (A). Putative salt-bridging interactions indicated by dashed ovals. (C) Density for CoQ-10 binding in the proximal Q_N-site from the CIII₂ focused maps. CIII₂ subunits are in green, and CoQ, heme b_H, and the side chain of MT-CYB His201 are shown as sticks and colored by atom, with carbon gray, nitrogen blue, oxygen red, and iron orange. (D) Density for CoQ-10 binding in the distal Q_N site from the CIII₂ focused maps. CIII₂ subunits, CoQ, heme b_H, and the MT-CYB side chain His201 are shown as in (C). (E) Density for CoQ binding in the distal Q_P site from the CIII₂ focused maps. CIII₂ subunits, CoQ, and heme b_L are shown as in (C). (F) Slice through the membrane domains of CI and CIII₂ within the closed structure of the SC looking from the mitochondrial matrix. Amphipol-lipid belt is shown at low contour in red, and CI (blue) and CIII₂ (green) are shown at a higher contour. Each CIII protomer is colored a different shade of green. CoQ active sites are marked: solid wedges for location on the matrix leaflet of the membrane and dashed wedges for inter-membrane space leaflet. Black and yellow ovals indicate lipid binding pockets of CI; black and yellow circle indicates lipid binding pocket of NDUFA11; black and yellow X indicates barrier to CoQ diffusion caused by contact site between CI and CIII₂ in the membrane. Yellow arrow illustrates the shortest path for CoQ diffusion from the CI Q-tunnel to the proximal Q_P site of CIII₂.

Figure 7

Local Resolution Maps of the SC I+III₂ Reconstructions Local resolution maps of the three 4.2 Å SC I+III₂ structures shown with the closed state (left), open state 1 (middle), and open state 2 (right) throughout. (A) CI side view. (B) Slice through CI from the same view as in (A). (C) View from the mitochondrial matrix. (D) Slice through the membrane domains of CI and CIII₂ from the same view as in (C). (E) Slice through the membrane domains of CI and CIII₂ viewed from the “heel” of CI looking at the position indicated by the dashed lines in (C). See also Figure S7.