Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction.

We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya, T., et al. (2014) Biochemistry 53, 7816-7823]. This two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme. However, because the reactivities of ring-strained cycloalkynes are generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We found that instead of strain-promoted click chemistry, Diels-Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine is more efficient for the pinpoint modification. An excess of quinone-site inhibitors did not interfere with Diels-Alder cycloaddition between the cyclopropene and tetrazine. These results along with the previous findings (cited above) strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single-particle cryo-electron microscopic studies, the channel is open or undergoes large structural rearrangements to allow bulky ligands into the proximity of 49 kDa Asp160.