Cytochrome b 560 ( QPs 1 ) of Mitochondrial Succinate - Ubiquinone Reductase

Mitochondrial succinate-ubiquinone reductase is composed of two parts, a water-soluble succinate dehydrogenase and a two-polypeptide membrane-anchoring protein fraction (QPs). The larger polypeptide of QPs is believed to be associated with cytochrome bas, (QPsl). The structure of QPsl was studied by immunochemistry and molecular cloning and sequencing. Antibodies against QPsl were raised in rabbits, purified, and characterized by enzyme-linked immunosorbent assay and Western blotting. The purified antibodies inhibited 75% of the reconstitutive activity of QPs and reacted with both submitochondrial particles (SMP) and mitoplasts. The binding of these antibodies to SMP was greatly increased when succinate dehydrogenase was removed from SMP by alkaline treatment, indicating that QPsl is a transmembranous protein and that some of its specific epitopes are covered by succinate dehydrogenase. Anti-QPsl antibodies were used to screen one cDNA clone encoding QPsl from a bovine heart cDNA Xgtll expression library. The cDNA insert is 946 base pairs with an open reading frame of 396 base pairs that encodes for 132 amino acid residues. The molecular weight of QPsl, calculated from the deduced amino acid sequence, is 14,320. Although the apparent molecular weight of QPsl, estimated by high resolution SDS-polyacrylamide gel electrophoresis, is 11,000, the existence of a presequence was ruled out by mass spectrometric analysis of protein fragments. QPsl is a very hydrophobic protein. Three probable membranespanning segments were revealed by a hydropathy plot of the sequence. QPsl has a higher sequence similarity to the sdhC peptide of Escherichia coli than to the sdhC peptide (cytochrome baas) of Bacillus subtilis. Like the bacterial proteins, QPsl has 2 conserved histidines at positions 34 and 90. The conserved nature and similar location of these 2 histidines, on the matrix-side surface of the membrane, suggest that they are involved in heme ligation of cytochrome beeo.