Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling

AbstractNADH:ubiquinone oxidoreductase (complex I) is the first, largest and most complicated enzyme of the mitochondrial electron transport chain. Photoaffinity labeling with the highly potent and specific inhibitor trifluoromethyldiazirinyl-[3H]pyridaben ([3H]TDP) labels only the PSST and ND1 subunits of complex I in electron transport particles. PSST is labeled at a high-affinity site responsible for inhibition of enzymatic activity while ND1 is labeled at a low-affinity site not related to enzyme inhibition. In this study we found, as expected, that 13 complex I inhibitors decreased labeling at the PSST site without effect on ND1 labeling. However, there were striking exceptions where an apparent interaction was found between the PSST and ND1 subunits: preincubation with NADH increases PSST labeling and decreases ND1 labeling; the very weak complex I inhibitor 1-methyl-4-phenylpyridinium ion (MPP+) and the semiquinone analogue stigmatellin show the opposite effect with increased labeling at ND1 coupled to decreased labeling at PSST in a concentration- and time-dependent manner. MPP+, stigmatellin and ubisemiquinone have similarly positioned centers of highly negative and positive electrostatic potential surfaces. Perhaps the common action of MPP+ and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I