Identification of the ubiquinone-binding site of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

In order to localize the ubiquinone-binding site of complex I (NADH:ubiquinone oxidoreductase), a novel photoreactive ubiquinone analogue (Q0C7ArN3) has been synthesized. It is shown that the direct chemical precursor of this analogue (Q0C7ArNO2) and the analogue itself are accepted as substrates in an enzyme assay utilizing ubiquinone-depleted mitochondrial membranes of Neurospora crassa. The activity of the enzyme applying these derivatives is inhibited by 50% at a concentration of 9 and 20 microM rotenone. Photoaffinity labeling experiments were performed with both isolated complex I and whole mitochondrial membranes of N. crassa under various conditions. In each of these experiments a protein subunit with an apparent molecular mass of about 9.5 kDa was labeled with high specificity. Radioactive labeling was totally prevented by the addition of ubiquinone-2 at concentrations higher than 500 microM but was not affected by comparable concentrations of rotenone or other hydrophobic substances. In the labeling experiments using whole membranes, the labeling signal was dramatically increased in the presence of 1.5 mM NADH. These results strongly suggest that the ubiquinone analogue interacts specifically with the enzyme.