Semidehydroascorbate as a product of the enzymic conversion of dopamine to norepinephrine. Coupling of semidehydroascorbate reductase to dopamine-beta-hydroxylase.

Abstract

Dopamine-β-hydroxylase, a copper monooxygenase, catalyzes the ascorbate-dependent hydroxylation of dopamine to norepinephrine. In the transfer of two electrons from ascorbate to dopamine-β-hydroxylase, the immediate product of enzyme reduction is not dehydroascorbate, as previously thought, but rather semidehydroascorbate, a free radical. Using the NADH-dependent semidehydroascorbate reductase preparation from rat liver microsomes, generation of semidehydroascorbate during dopamine-β-hydroxylation was observed. In the presence of catalytic amounts of ascorbate, a coupling of dopamine-β-hydroxylase to semidehydroascorbate reductase has been demonstrated: Regeneration of active cofactor is observed by the catalytic oxidation of NADH and formation of β-hydroxylated product; delayed addition of the reductase results in a decrease in the rate of NADH oxidation; and, with further delay, no oxidation of NADH is observed, indicating the lability of the free radical intermediate. At low rates of hydroxylation, a stoichiometric oxidation of NADH was obtained in the presence of the reductase during the formation of octopamine from tyramine. Semidehydroascorbate reductase was found in membrane fractions of the bovine adrenal medulla but not in the soluble fractions. Tyramine-dependent oxidation of NADH, inhibited by fusaric acid and stimulated by fumarate, can be observed using partially purified chromaffin vesicle membranes or large granular fraction membranes. These results are consistent with a role for semidehydroascorbate reductase in dopamine-β-hydroxylation through regeneration of the active cofactor, ascorbate.