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.