Work

Mycothiazole enables structural characterization of entire ubiquinone-accessing tunnel in mitochondrial respiratory complex I

Year: 2026

Type: preprint

Abstract: Mitochondrial NADH-ubiquinone (UQ) oxidoreductase (respiratory complex I) reduces UQ within its long tunnel-like cavity, which is coupled to triggering proton translocation at the membrane domain. Although mammalian complex I adopts distinct “open” and “closed” conformations, the architecture of the UQ-accessing tunnel in the open conformation remains poorly defined owing to partial disorder of the UQ catalytic region near Fe-S cluster N2 in the tunnel. Here, we applied cryo-electron microscopy (EM) single particle analysis to determine the structures of complex I in bovine heart submitochondrial particles in the presence or absence of a mycothiazole-type inhibitor. Preceding photoaffinity labeling experiments using a synthetic photoreactive derivative ([ 125 I]pMT) revealed that mycothiazole specifically labels the 49-kDa and ND1 subunits, both of which form a part of the UQ-accessing tunnel. Cryo-EM analysis demonstrated that bound mycothiazole reorganizes and stabilizes the UQ catalytic region in both open and closed conformations, enabling the first complete structural modeling of the entire UQ-accessing tunnel in the open conformation. Unexpectedly, in the presence of bound mycothiazole, loops of the PSST subunit forming the catalytic region in the tunnel adopted closed conformation-like features, whereas open conformation-like features were determined for the transmembrane helices of ND1 and ND6 subunits. The present study revealed that bound mycothiazole can reorganize the UQ catalytic region in the UQ-accessing tunnel and lead to a hybrid conformational state beyond the canonical open– versus –closed conformational model. Significance Statement Respiratory complex I is a central energy-converting enzyme that couples ubiquinone (UQ) reduction to proton translocation across the inner mitochondrial membrane. Mammalian complex I adopts distinct open and closed conformations, but the functional significance of the open conformation remains unresolved. Structural characterization of the UQ-reduction region in the open conformation has been limited by disorder of key elements surrounding the catalytic site. Using mycothiazole as a molecular probe, we stabilized and visualized the entire UQ-accessing tunnel in the open conformation. Unexpectedly, mycothiazole reorganized the catalytic region in both conformations into a common hybrid architecture, revealing that structural elements around the UQ catalytic site and those linked to proton translocation can rearrange independently.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.01.756109