Purification and characterization of highly purified cytochrome b from Complex III of baker's yeast

A simple, high-yield purification procedure for cytochrome b from yeast Complex III has been developed. This procedure involves solubilization using chemical modification of the lysine residues with 3,4,5,5-tetrahydrophthalic anhydride followed by hydroxyapatite column chromatography. This purified cytochrome b has a heme content of 37.0 nmol cytochrome b/mg and a molecular weight on SDS gels of 25000–26000. Amino acid analysis indicates high hydrophobicity and is very comparable to the composition deduced from the gene sequence (Nobrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828–9837). The latter data indicate a molecular weight of 42000 for the polypeptide; our heme analyses thus imply the presence of two hemes per polypeptide chain. Optical and MCD spectra are typical of a low-spin b-type cytochrome. MCD-potentiometric titration indicates a one-electron carrier with a single midpoint potential of ?44 mV at pH 7.4 and 25°C. The EPR spectrum of isolated cytochrome b has only one g_z signal at 3.70, indicating that the ‘strained’ heme structure (Carter, K., T'sai, A. and Palmer, G. (1981) FEBS Lett. 132, 243–246) is still maintained. No indication of antimycin binding was demonstrated either by the direct-fluorescence method or binding-precipitation method although stoichiometric binding to the parent Complex III was readily demonstrated.