Triton X-100 as a specific inhibitor of the mammalian NADH-ubiquinone oxidoreductase (Complex I)

Triton X-100 inhibits the NADH oxidase and rotenone-sensitive NADH-Q(1) red uctase activities of bovine heart submitochondrial particles (SMP) with an apparent K-i of 1 x 10(-5) M (pH 8.0, 25 degrees C). The NADH-hexammineruth enium reductase, succinate oxidase, and the respiratory control ratio with succinate as the substrate in tightly coupled SMP are not affected at the i nhibitor concentrations below 0.15 mM. The succinate-supported aerobic reve rse electron transfer is less sensitive to the inhibitor (K-i = 5 x 10(-5) M) than NADH oxidase. Similar to rotenone, limited concentrations of Triton X-100 increase the steady-state level of NAD(+) reduction when the nucleot ide is added to tightly coupled SMP oxidizing succinate aerobically, Also s imilar to rotenone, Triton X-100 partially protects Complex I against the t hermally induced deactivation and partially activates the thermally deactiv ated enzyme. The rate of the NADH oxidase inhibition by rotenone is drastic ally decreased in the presence of Triton X-100 which indicates a competitio n between these two inhibitors for a common specific binding site. In contr ast to rotenone, the inhibitory effect of Triton X-100 is instantly reverse d upon dilution of the reaction mixture. The NADH-Q(1) reductase activity o f SMP is inhibited non-competitively by added Q(1) whereas a simple competi tion between Q(1) and the inhibitor is seen for isolated Complex I. The res ults obtained show that Triton X-100 is a specific inhibitor of the ubiquin one reduction by Complex I and are in accord with our previous findings whi ch suggest that different reaction pathways operate in the forward and reve rse electron transfer at this segment of the mammalian respiratory chain. ( C) 1999 Elsevier Science B.V. All rights reserved.