Site-Specific Chemical Labeling of Mitochondrial Respiratory Complex I through Ligand-Directed Tosylate Chemistry

Abstract

The site-specific chemical modification of NADH-quinone oxidoreductase (complex I) by various functional probes such as fluorophores and microbeads, without affecting the enzyme activity, may allow single-molecule analyses of putative dynamic conformational changes in the enzyme. In an attempt to address this challenge, we performed site-specific alkynylation of complex I in bovine heart submitochondrial particles by means of a ligand-directed tosylate (LDT) chemistry strategy with synthetic acetogenin ligand 1, which has an alkynylated tosylate in the tail moiety, as a high-affinity ligand against the enzyme. The terminal alkyne was chosen as the tag to be incorporated into the enzyme because this functional group can serve as a "footing" for subsequent diverse chemical modifications via so-called click chemistry (i.e., azide-alkyne [3+2] cycloaddition in water). To identify the position alkynylated by ligand 1, fluorescent tetramethylrhodamine was covalently attached to the incorporated alkyne by click chemistry after the solubilization of complex I. Detailed proteomic analyses revealed that alkynylation occurred at Asp160 in the 49 kDa subunit, which may be located in the inner part of the putative quinone-binding cavity. The alkynylation was completely suppressed in the presence of an excess of other inhibitors such as bullatacin and quinazoline. While the reaction yield of the alkynylation step via LDT chemistry was estimated to be ~50%, the alkynylation unfortunately resulted in the almost complete inhibition of enzyme activity. Nevertheless, the results of this study demonstrate that complex I can be site-specifically alkynylated through LDT chemistry, providing a clue about the diverse chemical modifications of the enzyme in combination with click chemistry.

Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I

“…Some molecular tags, such as fluorophore and biotin, can be covalently attached to the terminal acetylene via Cu +1 -catalyzed click chemistry following photoaffinity labeling, which enables purification and/or detection of the labeled peptides. 18) We confirmed that PRA1 and PRA2 maintain the potent inhibitory activities (Table 2), and are the most potent class in this study. Photoaffinity labeling experiments are currently under way in our laboratory using these photoreactive amilorides.…”

Redox-induced activation of the proton pump in the respiratory complex I

“…Asp-139 resides on a four-helix bundle, which is known to be conformationally flexible based on structural and inhibitorbinding studies (12,23). A likely reason for the observed behavior is the abstraction of the proton from the Asp-139/His-38 ion pair that links to the Q reduction.…”

Identification of the Binding Position of Amilorides in the Quinone Binding Pocket of Mitochondrial Complex I

“…Alkynylated protein was identified and characterized using the Bruker Autoflex III 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 9 equipped with Ultimate 3000 nano-LC (LC-MS, Thermo Scientific), respectively, under the same experimental and data-processing conditions as those previously reported (15,16). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 10…”

“…In order to visualize the incorporated alkyne, a fluorescent TAMRA-azido tag was conjugated with the alkyne via Cu +1 -catazyzed click chemistry (15). As shown in Figure 3, a fluorescent band was exclusively observed at ~50 kDa, indicating that the terminal alkyne was successfully transferred from AAT to the ~50 kDa protein.…”