Reductive inactivation of the mitochondrial three subunit NADH dehydrogenase.

The 3-subunit iron-sulfur flavoprotein (NADH-artificial electron acceptor oxidoreductase) derived from complex I (EC 1.6.5.3) is rapidly and irreversibly inactivated in the presence of NADH. The rate of inactivation increases with a decrease of the enzyme concentration. The activities with ferricyanide, menadione and cytochrome c were lost synchronously during preincubation of the enzyme in the presence of NADH or dithionite under either aerobic or anaerobic conditions. The titration of the inactivation rate with the NADH/NAD+ pair suggests that reduction of a component with Em' = -325 mV (n = 2) is a prerequisite for a loss of the enzyme activity. Among the compounds tested only FMN and NAD+ were able to protect the enzyme against the reductive inactivation. NADH-induced loss of the enzyme activity in diluted solutions is accompanied with the synchronous appearance of a fluorescence characteristic for free FMN. It is concluded that the reduction of flavin leads to a strong decrease of FMN affinity to its specific binding site, and possible implications of the redox-dependent affinity changes in operation of NADH-ubiquinone reductase are discussed.