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-[(3)H]pyridaben ([(3)H]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.
Schuler
F
F
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, 115 Wellman Hall, Berkeley, CA 94720-3112, USA.
Casida
J E
JE
D015655:1-Methyl-4-phenylpyridinium / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006358:Hot Temperature; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019920:Photoaffinity Labels; D011090:Polyenes / Q000737:chemistry / Q000494:pharmacology; D011724:Pyridazines / Q000494:pharmacology; D012402:Rotenone / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014316:Tritium; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D019920:Photoaffinity Labels; D011090:Polyenes; D011724:Pyridazines; D012402:Rotenone; D009243:NAD; D014316:Tritium; D014451:Ubiquinone; C428725:pyridaben; C060262:2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone; C041573:stigmatellin; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; D015655:1-Methyl-4-phenylpyridinium