Photoaffinity labelling of mitochondrial NADH dehydrogenase with arylazidoamorphigenin, an analogue of rotenone

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Abstract
A photoaffinity-labeling analog of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analog inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabeled analog bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of MW 33,000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabeling by the analog and their sensitivity to rotenone, providing further evidence that the 33,000-MW protein forms part of the rotenone-binding site.