Dissected antiporter modules establish minimal proton-conduction elements of the respiratory complex I

Abstract

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts in recent decades, its long-range energy transduction principles remain highly debated. We create here minimal proton-conducting membrane modules by engineering and dissecting the key elements of the bacterial Complex I. By combining biophysical, biochemical, and computational experiments, we show that the isolated antiporter-like modules of Complex I comprise all functional elements required for conducting protons across proteoliposome membranes. We find that the rate of proton conduction is controlled by conformational changes of buried ion-pairs that modulate the reaction barriers by electric field effects. The proton conduction is also modulated by bulky residues along the proton channels that are key for establishing a tightly coupled proton pumping machinery in Complex I. Our findings provide direct experimental evidence that the individual antiporter modules are responsible for the proton transport activity of Complex I. On a general level, our findings highlight electrostatic and conformational coupling mechanisms in the modular energy-transduction machinery of Complex I with distinct similarities to other enzymes.

Fig. 1. Structure and function of complex I.

a Reduction of quinone (Q) triggers proton pumping in the membrane domain of Complex I. The subunit naming is based on Thermus thermophilus Complex I. b Closeup of antiporter-like subunits, Nqo12 (left, lateral helix not shown) and Nqo13 (right). Conserved residues along the hydrophilic axis are shown, and the trans-membrane broken helices TM7a/b and TM12a/b are highlighted in solid colour.

Fig. 2. Protonation pathways and modulation of proton transfer barriers.

a Proton pathways from MD simulations of Nqo12^ΔTH. Top inset: MD snapshot showing water-mediated connectivity from Lys329 to Lys385 and conserved polar residues stabilising the proton pathways. Bottom inset: The Glu132/Arg163 - Asp166/Lys216 ion-pair conformation in the WT (cyan) and E132Q (orange) variants. b Proton pathways from MD simulations of Nqo13. Top inset: MD snapshot of water-mediated connectivity from Lys235 to Glu377 via His292, and conserved polar residues stabilising the proton pathway. Bottom inset: the Glu123/Lys204 ion-pair conformation in WT (cyan) and E123Q (orange) variants. c, e Ion-pair dynamics and d, f hydration level along the proton pathways in the WT and E132Q/E123Q variants of Nqo12^ΔTH (c, d) and Nqo13 (e, f); symbols in (d, f) refer to the pathway region indicated in panel (a) and (b), respectively. g, i Free energy profiles of proton transfer along the proton channel in the WT (blue) and E132Q (E123Q, orange) variants of (g) Nqo12^ΔTH and (i) Nqo13. h, j Modulation of proton transfer barrier with ion-pair distance based on QM/MM calculations (black dots) and a fit from a theoretical model (see Supplementary Information). Inset: dissociation of the ion-pair induces an electric field (green dots) along the proton pathways. Data are provided in the Source Data file.

Fig. 3. Biophysical characterisation of proton conduction in dissected antiporter-like modules induced by addition of weak acid.

a Proteoliposome assay for probing the proton conduction kinetics in the dissected antiporter-like subunits with pyranine (HPTS) by addition of K⁺CH₃COO^-. Addition of 10 mM acetate to (b) Nqo12^ΔTH- and (c) Nqo13- proteoliposomes induced conduction of protons across the membrane. The ionophore nigericin was added to dissipate the generated ∆pH. The final steady-state ∆pH level before addition of nigericin is shown for the (d) Nqo12^ΔTH and (e) Nqo13 constructs. The protonation conduction rate is sensitive to substitution of the conserved ion-pair and residues along the proton pathway. The data is compared to proton conduction assayed with empty liposomes (EL, n = 3) and aquaporin (AqpZ) reconstituted proteoliposome. Note that the initial pH gradient pre-equilibrates upon mixing the proteoliposomes in the buffer, leading to a higher initial baseline for the fast-conducting constructs (E123Q¹³, n = 3/ E132Q¹²), while the final steady-state pH (d, e), obtained prior to addition of nigericin, is independent of the baseline. Data shown are derived from independent experiments where n = 6 (mean ± SD) unless specified differently. See Supplementary Fig. 11 for addition of weak base, and Supplementary Fig. 13b, c for ∆pH and Δψ-mediated proton transport. Data are provided in the Source Data file.

Fig. 4. Biophysical characterisation of proton conduction properties in dissected antiporter-like modules co-reconstituted with F₁F_o-ATP synthase.

a Proteoliposome assay for probing the proton conduction kinetics in the dissected antiporter-like subunits co-reconstituted with ATP synthase, monitored by fluorescence quenching of ACMA. Addition of 0.2 mM ATP generates an ATPase-driven ∆pH across the proteoliposome membrane, which competes with the (d) Nqo12 ^ΔTH- and (f) Nqo13-mediated proton transport. b, c Structure of the proton pathways from MD simulations of the WT (blue) and ion-pair mutants (orange) for (b) Nqo12^ΔTH and (c) Nqo13. Sidechains of conserved residues are shown as sticks, water molecules as spheres. Relative ACMA quenching amplitudes for co-reconstituted ATP synthase with (e) Nqo12^∆TH and (g) Nqo13 constructs. Data shown are derived from independent experiments where n = 6 (mean ± SD), except F₁F_o-K235M¹³ where n = 5. Data are provided in the Source Data file.

Fig. 5. Mechanistic model of proton transport in the antiporter modules.

Schematic model of (a) antiporter-mediated proton transport. Blue/grey lines schematically show conneted/disconnected proton wires, and red dotted line an electrostatic repulsion. Blue/red/white circles – putative positive/negative/neutral protonation states. pK_a^A – pK_a of N-side proton acceptor; pK_a^D – pK_a of P-side proton donor; ΔG^O→C – free energy closing ion pair; pH_out – pH of N-side; pH_in – pH of P-side. b Comparison of a proton pumping model in the intact Complex I.