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Pinpoint Dual Chemical Cross-Linking Explores the Structural Dynamics of the Ubiquinone Reaction Site in Mitochondrial Complex I.

The ubiquinone reduction step in NADH-ubiquinone oxidoreductase (complex I) is the key to triggering proton translocation in its membrane part. Although the existence of a long and narrow quinone-access channel has been identified, it remains debatable whether the channel model can account for binding of various ligands (ubiquinones and inhibitors) to the enzyme. We previously proposed that the matrix-side interfacial region of the 49 kDa, ND1, PSST, and 39 kDa subunits, which is covered by a loop connecting transmembrane helices (TMHs) 1 and 2 of ND3, may be the area for entry of some bulky ligands into the quinone reaction cavity. However, this proposition lacks direct evidence that the cavity is accessible from the putative matrix-side region, which allows ligands to pass. To address this, we examined whether Cys39 of ND3 and Asp160 of 49 kDa can be specifically cross-linked by bifunctional cross-linkers (tetrazine-maleimide hybrid, named TMBC). On the basis of the structural models of complex I, such dual cross-linking is unexpected because ND3 Cys39 and 49 kDa Asp160 are located on the TMH1-2 loop and deep inside the channel, respectively, and hence, they are physically separated by peptide chains forming the channel wall. However, three TMBCs with different spacer lengths did cross-link the two residues, resulting in the formation of new cross-linked ND3/49 kDa subunits. Chemical modification of either ND3 Cys39 or 49 kDa Asp160 blocked the dual cross-linking, ensuring the specificity of the cross-linking. Altogether, this study provides direct evidence that the quinone reaction cavity is indeed accessible from the proposed matrix-side region covered by the ND3 TMH1-2 loop.