STRUCTURAL BASIS FOR THE ELECTRON-TRANSFER ACROSS THE CHROMAFFIN VESICLE MEMBRANES CATALYZED BY CYTOCHROME B(561) - ANALYSES OF CDNA NUCLEOTIDE-SEQUENCES AND VISIBLE ABSORPTION-SPECTRA

We isolated cDNA clones for cytochromes b(561) 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 ful ly-conserved regions in this cytochrome. In addition, one methionyl an d 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 ((69)ALLVYRVFR(77)) is located on the extravesicul ar side of an alpha-helical segment and the second one ((SLHSW124)-S-1 20) 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 seque nces are likely to form the binding sites for extravesicular ascorbic acid and intravesicular semidehydroascorbic acid, respectively. A mild alkaline-treatment of purified bovine cytochrome b(561) in oxidized s tate led to a specific loss of an electron-accepting ability from asco rbic acid for a half of the heme center, suggesting a distinct role fo r each of the two hemes. (C) 1998 Elsevier Science B.V.