A highly active ubiquinol-cytochrome c reductase (bc1 complex) from the colorless alga Polytomella spp., a close relative of Chlamydomonas. Characterization of the heme binding site of cytochrome c1.
The alga Polytomella spp. offers extraordinary advantages in the preparation of mitochondria since it lacks chloroplasts and a cell wall. In this work the mitochondrial bc1 complex from Polytomella spp. was solubilized and purified by ion exchange chromatography. The complex was found to be composed of 10 polypeptides and exhibited high rates of ubiquinol-cytochrome c oxidoreductase activity (> 300 s-1) sensitive to antimycin and myxothiazol. The molecular mass of the bc1 complex from Polytomella spp. was assayed by gel filtration and estimated to be of 256,300 Da. Therefore, this complex exhibits the unique property of behaving as a monomer. Amino-terminal sequencing of cytochrome c1 identified 7 residues, from which a deoxyoligonucleotide was designed. A second deoxyoligonucleotide was constructed based on a highly conserved region of the c1 type cytochromes. With these probes, a fragment of the cytochrome c1 gene was amplified by polymerase chain reaction and sequenced. The deduced sequence of the apoprotein exhibited a consensus binding site CXXCH. The data suggest that the cytochrome c1 from Polytomella spp. differs from other protoctists like Crithidia and Euglena, i.e. it exhibits a heme binding domain structurally related to the bovine, yeast, and Neurospora c1 type cytochromes.
Gutiérrez-Cirlos
E B
EB
Departamento de Bioenergética, Universidad Nacional Autónoma de México, Distrito Federal.
Antaramian
A
A
Vázquez-Acevedo
M
M
Coria
R
R
González-Halphen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003575:Cytochromes c1 / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D014450:Electron Transport Complex III / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D056890:Eukaryota / Q000201:enzymology*; D006418:Heme / Q000737:chemistry; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D016823:Volvocida / Q000201:enzymology*
D017931:DNA Primers; D015800:Protozoan Proteins; D006418:Heme; D003575:Cytochromes c1; D014450:Electron Transport Complex III