Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes.

Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. The midpoint redox potential of the cytochrome is 100-130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5-7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. The Nernst plots of titration data have slopes of 75-115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4-6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.