NONHEME IRON PROTEIN AS A POSSIBLE SITE OF ROTENONE INHIBITION OF MITOCHONDRIAL NADH DEHYDROGENASE

The inhibition by rotenone of NADH oxidase activity of submitochondrial particles (ETP) has been studied, and a titer of 0.07 mμmoles rotenone/mg protein has been found for 50% inhibition. The extent of reduction of flavoproteins has been examined spectrophotometrically using the wavelength pair 470 minus 500 nm, during the slow oxidation of NADH in the presence of varying concentrations of rotenone. With concentrations of rotenone of 0.2 mμmoles/mg protein or more, a pigment was observed which showed a decrease in absorbance at 470 nm relative to 500 nm. This pigment remains reduced after the oxidation of NADH by the rotenone inhibited system. The optical difference spectrum of this pigment is characterized by two broad troughs with minima at about 415 and 455 nm. The extent of reduction of the nonheme iron protein associated with NADH dehydrogenase has been evaluated by electron paramagnetic resonance spectroscopy. These experiments showed the presence of an EPR signal at g = 1.94 after the slow oxidation of NADH in the presence of rotenone. From these results it is suggested that the nonheme iron protein associated with NADH dehydrogenase is involved in the mechanism of inhibition of NADH oxidation by rotenone. © 1969.