Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization

Abstract

Spectrophotometric and fluorimetric substrate couple titrations and potentiometric spectrophotometric titrations were used to determine the oxidation-reduction potentials of components showing absorbance or fluorescence at the wavelengths attributable to the flavoproteins of mitochondria fractionated using digitonin together with sonication. A pure mitoplast fraction devoid of cytochrome b5 contamination could be obtained using 230 micrograms digitonin/mg of mitochondrial protein. The digitonin-soluble fraction contained a species having Em7.4 = -123 mV and probably represents the outer membrane flavoproteins. The inner membrane-matrix fraction, treated with ultrasound, provided evidence of a flavoprotein species with redox potential (Em7.4 = -302 mV) in the matrix fraction. The -302 mV component is probably lipoamide dehydrogenase. A high redox potential species with Em7.4 = +19 mV in titrations with the succinate fumarate couple was located in the inner membrane vesicles and is probably identical with succinate dehydrogenase. The electron-transferring flavoprotein (ETF) was isolated from bovine heart mitochondria and its Em7.4 = -74 mV determined. The component in the matrix fraction with an apparent Em7.4 = -56 mV probably represents ETF, and that in the inner membrane fraction with an apparent Em7.4 = -43 mV the NADH dehydrogenase flavoprotein. A component in an apparently low concentration with Em7.4 = +30 mV was detected in the inner membrane fraction. This probably represents the ETF-dehydrogenase flavoprotein. The origin of the flavoprotein fluorescence of mitochondria and intact tissues is discussed.