Respiratory Complex I (NADH:ubiquinone oxidoreductase) is the largest enzyme in the respiratory chain. It transfers two electrons from NADH to ubiquinone (UQ), coupled with the translocation of four protons across the membrane. Complex I dysfunction is implicated in numerous diseases. The bacterial enzyme is a simpler model, consisting of 14 core subunits, compared to the more complex mitochondrial enzyme. Previous research has provided structural information via X-ray crystallography and cryo-EM, but the mechanism coupling electron transfer to proton translocation remains unclear. This study aims to elucidate this mechanism by analyzing high-resolution structures of Complex I in different conformational states, particularly focusing on the impact of quinone binding.

Extensive research has been conducted on Complex I's structure and function. Walker (1992) provided early insights into its structure. Yagi and Matsuno-Yagi (2003) highlighted its proton-translocating capability. Brandt (2006) and Hirst (2013) summarized its structure and mechanism. Sazanov (2015) reviewed its structure and function, proposing a conformational mechanism. Studies on respirasomes revealed Complex I's involvement in supercomplexes (Letts et al., 2016; Guo et al., 2017; Letts & Sazanov, 2017). Numerous disease-related mutations in its 45 subunits have been identified (Smeitink et al., 2001; Koopman et al., 2013; Fiedorczuk & Sazanov, 2018). Prior structural studies using crystallography and cryo-EM (Sazanov & Hinchliffe, 2006; Sazanov, 2007; Berrisford & Sazanov, 2009; Efremov & Sazanov, 2011; Baradaran et al., 2013; Fiedorczuk et al., 2016; Hunte et al., 2010; Zickermann et al., 2015; Zhu et al., 2016; Agip et al., 2018) have laid the groundwork for understanding its structure. However, the crucial link between redox energy and proton translocation requires further investigation.

This study used a multi-pronged approach: **Complex I Purification:** The researchers purified Complex I from *T. thermophilus* using various chromatography techniques (ion exchange, gel filtration). Subunit Nqo16 was also purified separately and later added to improve crystallization efficiency. **Activity Measurements:** Complex I activity was measured using NADH as an electron donor and decylubiquinone (DQ) as an electron acceptor. Inhibitor studies were conducted to confirm complex functionality. **Crystallization:** Crystals of Complex I were obtained using sitting-drop vapor diffusion under various conditions. Crystals were soaked with NADH, DQ, and other quinone inhibitors (piericidin A, aureothin, pyridaben) to capture different conformational states. **X-ray Crystallography:** Data was collected from crystals at various synchrotrons and processed using XDS and XSCALE. Structures were refined using phenix.refine, with ligands modelled where appropriate. **Cryo-Electron Microscopy:** Purified Complex I (with or without NADH/NAD+) was prepared for cryo-EM. Micrographs were collected using a 300 kV FEI Krios TEM. Data processing involved motion correction, CTF correction, particle picking, 2D and 3D classification, and refinement using RELION 2.0. Models were built using MDFF and Rosetta. **Structural Analysis:** Structural analyses were performed using PyMOL, VMD, and ProSMART. Protein solvation was modeled using Dowser. Normal Mode Analysis (NMA) was performed with Bio3D, using ANM and HCA. Network analysis of allosteric couplings was done to identify functional groups. Coarse-grained molecular dynamics (CG-MD) simulations using Martini v2.2 model were employed to study dynamics and allosteric couplings.

The study revealed several key findings: 1. **Conformational Heterogeneity:** Complex I exists in at least two main conformational states (major and minor), observed via cryo-EM regardless of NADH/NAD+ presence. The major state is energetically more favorable. The minor state shows a more open conformation with a larger angle between the peripheral and membrane arms. 2. **Limited NADH Effect:** NADH binding alone did not induce significant large-scale conformational changes except for a flip of H384 after prolonged exposure. This suggests that NADH reduction might primarily play a role in local rearrangements rather than triggering global conformational changes. 3. **Quinone's Key Role:** Binding of DQ (and other quinone analogues) to the Q-site caused a much more significant rotation and tilt of the peripheral arm, alongside structural changes in the nearby proton channel (E-channel). This rotation and structural shift are far more prominent than changes caused by NADH binding. 4. **Pathway of Conformational Changes:** Ligand binding initiates a pathway of local conformational changes that propagate from the Q-site to the proton pumps (E-channel) via Nqo4, Nqo8, Nqo10, and Nqo11. The extent of these changes correlates with the ligand's binding strength, with DQ leading to the most pronounced changes. 5. **Normal Mode Analysis (NMA):** NMA identified two major domain motions: bending and rotation. Overlap analysis revealed that the transition to the DQ-bound state involves large-scale rotational movements, unlike transitions between unliganded states. Deformation analysis identified hinge regions essential for these transitions. 6. **Network Analysis:** Network analysis showed strong coupling between motions in the Q-site and the E-channel regions, especially in the DQ-bound state. This confirmed the allosteric coupling between quinone binding and changes in the E-channel. 7. **Coarse-grained MD Simulations:** CG-MD simulations further confirmed the strong coupling between Q-site and E-channel motions, with the strongest coupling observed in the state with charges transferred to the amino acids coordinating quinone. This highlighted the role of charge transfer in propagating conformational changes. 8. **Water Networks:** Analysis of water networks found that a water molecule is only present between E32 and E67 of Nqo11 in the DQ-bound structure, potentially facilitating proton transfer between the E-channel and Nqo14.

The findings address the central question of how electron transfer in the peripheral arm is coupled to proton translocation in the membrane domain. The observation that quinone binding, rather than NADH reduction, initiates the major conformational changes suggests a new understanding of the coupling mechanism. The propagated changes observed in the E-channel highlight the importance of quinone binding and chemistry in triggering these structural shifts. The limited impact of NADH alone suggests that it primes the complex but the significant effects of quinone binding trigger the actual conformational transitions linked to proton translocation. The study highlights the importance of both global conformational changes and local rearrangements in the catalytic cycle. The two different conformational states identified in *T. thermophilus* may correspond to the "open" and "closed" states previously described in mammalian Complex I, although further study is needed to confirm this correspondence precisely. This suggests a conserved mechanism across species.

This study demonstrates that quinone binding plays a crucial role in the coupling mechanism of respiratory Complex I. The observed conformational changes, propagating from the quinone-binding site to the proton channels, suggest that quinone chemistry, rather than solely redox reactions of FeS clusters, drives the process. Future research should investigate the detailed proton transfer mechanism involving the antiporter-like subunits, the role of the lipid membrane, and the effects of the full-length natural quinone substrate.

The study primarily focused on *T. thermophilus* Complex I, a simplified bacterial model. Although the findings are likely relevant to the mammalian enzyme, direct confirmation using mammalian Complex I requires further investigation. The high-resolution structures obtained provide insights into specific conformational states, but may not fully capture the dynamics of the complete catalytic cycle. Finally, the study did not explicitly model the lipid membrane, which could affect the structural and functional aspects of Complex I.