Structural basis for the electron transfer across the chromaffin vesicle membranes catalyzed by cytochrome b561: analyses of cDNA nucleotide sequences and visible absorption spectra.
We isolated cDNA clones for cytochromes b561 from sheep and porcine adrenal medullae using the RT-PCR technique. Comparison of the deduced amino acid sequences of various species showed that there are two fully-conserved regions in this cytochrome. In addition, one methionyl and six histidyl residues (potential heme ligands) are fully-conserved. Based on a plausible structural model in which a polypeptide spans the vesicle membranes six times and holds two heme B molecules, the first conserved sequence (69ALLVYRVFR77) is located on the extravesicular side of an alpha-helical segment and the second one (120SLHSW124) is located in an intravesicular loop connecting two alpha-helical segments, respectively. Consideration of the relative locations of the fully-conserved sequences, and the methionyl and histidyl residues in the model led to a proposal that the first and second conserved sequences are likely to form the binding sites for extravesicular ascorbic acid and intravesicular semidehydroascorbic acid, respectively. A mild alkaline-treatment of purified bovine cytochrome b561 in oxidized state led to a specific loss of an electron-accepting ability from ascorbic acid for a half of the heme center, suggesting a distinct role for each of the two hemes.
Okuyama
E
E
Department of Biochemistry, Kagawa Medical University, Japan.
Yamamoto
R
R
Ichikawa
Y
Y
Tsubaki
M
M
D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism / Q000648:ultrastructure; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004579:Electron Transport; D008969:Molecular Sequence Data; D012756:Sheep; D013552:Swine
D003573:Cytochrome b Group; D018076:DNA, Complementary; C023646:cytochrome b561