The L-shaped architecture of complex I can be split into two segments, a membrane arm and a peripheral arm in which electrons are delivered from NADH via FMN and a series of ironsulfur clusters (FeS chain) to Q, which binds in a ~35 Å long tunnel (Figs. and). The head group of tunnel-bound Q resides at a site (called site 1 here, see Fig. ) within electron transfer distance from the N2 FeS cluster, which is the terminal FeS cluster of the FeS chain and donates electrons to Q one at a time. It is now understood that electron transfer to Q (and processes beyond it) drives the pumping of protons in the long membrane arm of complex I (Fig. ). The coupling between the Q redox-active site and the proton pumping site(s), which are maximally separated by ca. 200 Å, is known to be strong. For instance, point mutations in the membrane domain can perturb electron transfer activity in the hydrophilic peripheral domain of complex I . But how the coupling is achieved at a molecular level is a major unresolved issue in the field of bioenergetics. Several open-ended mechanistic questions remain. For instance, is it the full two-electron reduction of Q that is central to the coupling mechanism, or is the one-electron reduced semiquinone (SQ) state also important? At what stage does the protonation of reduced Q occurs and is this substrate proton transfer critical for pumping? How is proton transfer accomplished in the membrane domain of complex I? Next, we discuss several recent models of the coupling mechanism of complex I that attempt to answer these challenging questions through various experimental and computational approaches. . The core subunits of complex I are displayed by coloured ribbons, whereas the accessory subunits that surround the core subunits in transparent white surface. The redox active flavin mononucleotide (sticks) and iron-sulfur clusters (orange-yellow spheres) are shown together with the Q-binding tunnel (grey). The electron and proton transfers are depicted by dark orange and dark blue arrows, respectively. Location of two conserved acidic residues Glu107 (NDUFS2 subunit, green) and Glu39 (ND3 subunit, yellow) are marked with dotted back arrows (see also panel C). All amino acid numbering is based on Y. lipolytica complex I. (B) The ~35 Å long Q binding tunnel is shown in light grey with the Q binding sites numbered with respect to their proximity to the N2 cluster (orange-yellow spheres). Site 3 (not discussed in the text) is not marked but is present in the region between sites 2 and 4. The highly conserved active site residues of the NDUFS2 subunit (see text, sections 5 and 6) and E-channel residues (formed by residues Glu196, Glu231 and Glu147 of ND1 subunit, see text) are shown as sticks colored according to their respective subunits. The dark orange mesh is depicting the water occupancy in the Q binding tunnel and the E-channel , based on molecular dynamics (MD) simulations of complex I . The black star denotes the Q tunnel/E-channel junction, near the Q binding site 4, a putative PLS region (see text). (C) The two cavities that merge with the Q tunnel found by CAVER (17) using a minimal probe radius 0.9 Å and starting point from the sidechain oxygen atom of conserved Tyr144 from NDUFS2. The two key acidic residues Glu107 (NDUFS2) and Glu39 (ND3) are shown (see also panel A). Corresponding residues in mammalian complex I from bovine and E. coli bacterial complex I are Glu104/Glu236 and Glu38/Glu51, respectively. (D) The same two cavities, which may transfer protons (see text), fill with water molecules (shown as red spheres) in extended (3 µs) MD simulations of complex I . Water occupancy in panels B and D is shown at an isovalue of 0.2 (20% occupancy) by an orange mesh.