The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

Abstract

1. 1. Three b-type cytochromes ( b 557.5, b 560, and b 562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c 1, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major α and β absorption peaks of cytochromes b 557.5, b 560, b 562.5, c 1 and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles. 2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b 560, b 562.5 and c 1, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b 557.5 was found in Complex II (succinate-ubiquinone reductase). 3. 3. Cytochrome b 560 was readily reduced by NADH or succinate, but b 562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c 1 with ascorbate inhibited the subsequent reduction of b 562.5 by substrates. These results indicate that b 560 and b 562.5 correspond, respectively, to b K and b T previously described by Chance et al. 14 (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182). 4. 4. Similar to b 560, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c 1 inhibits its subsequent reduction by substrates. This property is similar to that of b 562.5. 5. 5. Cytochrome b 557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b 557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b 557.5 reoxidation by fumarate. 6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the α peaks of cytochrome c 1. The spectrum of cytochrome c 1 at 77 °K as modified by 3% (v/v) ethanol is shown.