DIPHENYLENEIODONIUM INHIBITS REDUCTION OF IRON-SULFUR CLUSTERS IN THEMITOCHONDRIAL NADH-UBIQUINONE OXIDOREDUCTASE (COMPLEX-I)

Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone o xidoreductase (Complex I) on the substrate side of the Fe-S clusters. In the inhibited NADH-supplemented state all of the Fe-S clusters are oxidized, whereas the reduced minus oxidized difference spectrum of th e protein-bound FMN can be visualized. It is characterized by troughs at 370 and 450 nm and a small increase of absorbance in the 500-700-nm region. DPI probably reacts irreversibly with FMN, because oxidation of FMN is blocked even after its extraction from the enzyme. Inhibitio n requires preincubation of enzyme in the presence of NADH and DPI. Th e lower the NADH/ NAD(+) ratio or the pH, or the higher the NAD(+)/DPI ratio, the more DPI is required for inhibition. NAD(+) and DPI appare ntly compete for a common site. Both ubiquinone and dichlorophenolindo phenol reductase activities are fully blacked by DPI, whereas the ferr icyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcom plex I lambda and the flavoprotein fraction. DPI also inhibits NADH ox idation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membr anes of Paracoccus denitrificans and Escherichia coli.