Synthesis of functional probes toward elucidation of the function of respiratory enzymes of Caenorhabditis elegans.

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To identify the binding subunit of fenpyroximate, a very potent inhibitor of bovine heart mitochondrial complex I, we synthesized ^3H-labeled photolabile fenpyroximate ([^3H](trifluoromethyl)-phenyldiazirinyl fenpyroximate, TDF). TDF appeared to elicit very potent inhibition of bovine heart mitochondrial complex I being nearly equivalent to that of fenpyroximate.On the basis of photoaffinity studies with bovine heart complex I in collaboration with Dr.Takao Yagi (The Scripps Research Institute, USA), the binding site of TDF was shown to be subunit ND5 which is thought to exist in membrane arm of the enzyme. Since antibody to the C-terminal region of the ND5 subunit only weakly recognized the subunit, we identified the ND5 by 2D-electrophoresis (SDS-PAGE). Our result is the first study wherein the subunit located in membrane arm was identified as the inhibitor binding subunit. Our study strongly suggests that subunit(s) located in membrane arm may be involved in the proton-pumping activity of complex I.