Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization.
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.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D004072:Digitonin; D004579:Electron Transport; D005260:Female; D005420:Flavoproteins / Q000302:isolation & purification / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D013050:Spectrometry, Fluorescence; D013367:Submitochondrial Particles / Q000378:metabolism
D005420:Flavoproteins; D004072:Digitonin