Pinpoint introduction of functional molecular probe into the NqrB subunit of Na+-translocating NADH-ubiquinone oxidoreductase from Vibrio cholerae

The Na⁺-translocating NADH-ubiquinone oxidoreductase (Na⁺-NQR) is a key enzyme in the respiratory chain of numerous pathogenic bacteria, including Vibrio cholerae. The flexible cytoplasmic N-terminal region of the NqrB subunit (Met¹–Lys⁵⁴), which may play an important role in the final UQ reduction at the adjacent NqrA, is the target of specific inhibitors. If we can develop a new method that enables pinpoint introduction of functional probe molecules (such as fluorescent probes) into the N-terminal region, this could lead to new experimental ways of monitoring dynamic structural changes of the region. We previously showed that an electrophilic chemical group, which can be released from korormicin A-templated synthetic ligand, can be predominantly introduced into nucleophilic Lys²² as a “foothold” via ligand-directed (LD) substitution, but the subsequent conjugation of a functional probe molecule to the foothold by Cu⁺-catalyzed click chemistry required destruction of the enzyme. Accordingly, we now report the nondestructive conjugation of the functional molecule into the N-terminal region via a two-step conjugation technique: first, pinpoint introduction of a foothold tag containing a ring-strained cyclopropene by LD substitution using a new korormicin A-templated ligand (BEK-1) and second, direct conjugation of a fluorescent probe molecule containing tetrazine with the introduced cyclopropene by inverse electron-demand Diels-Alder-type click chemistry. Protein sequence analyses revealed that the fluorescent probe is attached to Lys¹⁹, His²⁰, or Lys²² in the region. The extent of conjugation of the fluorescent probe was approximately halved in the presence of different inhibitors, suggesting that the inhibitor binding induces structural changes around the residues.