Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems.
Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000378:metabolism*; D002838:Chromaffin System / Q000378:metabolism*; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism*; D004579:Electron Transport; D007425:Intracellular Membranes / Q000378:metabolism; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D010105:Oxygenases / Q000378:metabolism*
D003573:Cytochrome b Group; D009097:Multienzyme Complexes; C023646:cytochrome b561; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D004299:Dopamine beta-Hydroxylase; C035189:peptidylglycine monooxygenase