Abstract
Diphenyleneiodonium (DPI) inhibits the mitochondrial NADH-ubiquinone oxidoreductase (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 the 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. Inhibition requires preincubation of enzyme in the presence of NADH and DPI. The 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 apparently compete for a common site. Both ubiquinone and dichlorophenolindophenol reductase activities are fully blocked by DPI, whereas the ferricyanide reductase activity is inhibited by 75%. Similar results were found with Complex I and two rotenone-insensitive preparations, subcomplex I lambda and the flavoprotein fraction. DPI also inhibits NADH oxidation by bacterial NADH-ubiquinone oxidoreductase-1 (NDH-1) in membranes of Paracoccus denitrificans and Escherichia coli.
Highlights
Diphenyleneiodonium(DPI)inhibitsthemitochoncatalyzes reduction of QI1and rotenone-insensitive reduction of drial NADH-ubiquinone oxidoreductase (ComplexI) on ferricyanide and dichlorophenolindophenol (DCIP).The Q1rethe substrate side of the Fe-S clusters
Inhibition requires incubation of the enzyme in the presence of NADH and DPI, and the sensitivity to DPI is dependent on the NADW NAD' ratio; this indicates that DPI reacts with a reduced enzyme
Its spectrum is similar to that previously reported for other flavoproteins (Ghisla et al, 1974)
Summary
Diphenyleneiodonium(DPI)inhibitsthemitochoncatalyzes reduction of QI1and rotenone-insensitive reduction of drial NADH-ubiquinone oxidoreductase (ComplexI) on ferricyanide and dichlorophenolindophenol (DCIP).The Q1rethe substrate side of the Fe-S clusters. We have studiedthe DPI inhibition of Complex I, subcomplex IA, the flavoprotein (FP)fraction, and the bacterial counterparts, NDH-1, from Paracoccus denitrificans and Escherichia coli (Yagi, 1991).Subcomplex IA contains about 15 polypeptide subunits, FMN, and at least four Fe-S clusters,and its Q1 reductase activity is insensitive to rotenone (Finel, 1993).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
