NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron-sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the l argest and least understood enzyme complex of the respiratory chain. The ma mmalian mitochondrial enzyme (also called complex: I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) i n Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 sub units, A major unsolved question is the location and mechanism of the termi nal electron transfer step from iron-sulfur cluster N2 to quinone, Potent i nhibitors acting at this key region are candidate photoaffinity probes to d issect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including ro tenone, piericidin A, bullatacin, and pyridaben, We designed (trifluorometh yl) diazirinyl [H-3] pyridaben ([H-3]TDP) as our photoaffinity ligand becau se it combines outstanding inhibitor potency, a suitable photoreactive grou p, and tritium at high specific activity. Photoaffinity labeling of mitocho ndrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity sp ecifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established p hotoaffinity labeling of the equivalent bacterial NQO6, Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the sp ecific high-affinity binding site of PSST is exceptionally sensitive to eac h of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conser ved inhibitor-binding site and that this subunit plays a key role in electr on transfer by functionally coupling iron-sulfur cluster N2 to quinone.