Publications

• Electron transfer in chromaffin-vesicle ghosts containing peroxidase.

  Harnadek G.J., Ries E.A., Tse D.G., Fitz J.S., Njus D.

  In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome ... >> More

  In chromaffin vesicles, the enzyme dopamine beta-monooxygenase converts dopamine to norepinephrine. It is believed that reducing equivalents for this reaction are supplied by intravesicular ascorbic acid and that the ascorbate is regenerated by importing electrons from the cytosol with cytochrome b-561 functioning as the transmembrane electron carrier. If this is true, then the ascorbate-regenerating system should be capable of providing reducing equivalents to any ascorbate-requiring enzyme, not just dopamine beta-monooxygenase. This may be tested using chromaffin-vesicle ghosts in which an exogenous enzyme, horseradish peroxidase, has been trapped. If ascorbate and peroxidase are trapped together within chromaffin-vesicle ghosts, cytochrome b-561 in the vesicle membrane is found in the reduced form. Subsequent addition of H2O2 causes the cytochrome to become partially oxidized. H2O2 does not cause this oxidation if either peroxidase or ascorbate are absent. This argues that the cytochrome is oxidized by semidehydroascorbate, the oxidation product of ascorbate, rather than by H2O2 or peroxidase directly. The semidehydroascorbate must be internal because the ascorbate from which it is formed is sequestered and inaccessible to external ascorbate oxidase. This shows that cytochrome b-561 can transfer electrons to semidehydroascorbate within the vesicles and that the semidehydroascorbate may be generated by any enzyme, not just dopamine beta-monooxygenase. << Less

  Biochim. Biophys. Acta 1135:280-286(1992) [PubMed] [EuropePMC]

• Mechanism of ascorbic acid regeneration mediated by cytochrome b561.

  Njus D., Kelley P.M., Harnadek G.J., Pacquing Y.V.

  In summary, ascorbic acid serves as a one-electron donor for dopamine beta-hydroxylase in chromaffin vesicles and probably for peptide amidating monooxygenase in neurohypophyseal secretory vesicles. It appears that the semidehydroascorbate that is produced is reduced by cytochrome b561 to regenera ... >> More

  In summary, ascorbic acid serves as a one-electron donor for dopamine beta-hydroxylase in chromaffin vesicles and probably for peptide amidating monooxygenase in neurohypophyseal secretory vesicles. It appears that the semidehydroascorbate that is produced is reduced by cytochrome b561 to regenerate intravesicular ascorbate. Cytochrome b561, a transmembrane protein, is reduced in turn by an extravesicular electron donor, probably cytosolic ascorbic acid. It will be interesting to see whether other ascorbate-requiring enzymes in other organelles use a similar ascorbate-regenerating system to provide an intravesicular supply of reducing equivalents. << Less

  Ann N Y Acad Sci 493:108-119(1987) [PubMed] [EuropePMC]