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

NADH 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-[H-3]pyridaben ([H-3]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. (C) 2001 Elsevier Science B.V. All rights reserved.