On Functional Role of Cytochrome b₅

II. NADH-linked Ascorbate Radical Reductase Activity in Microsomes

Abstract

1) Microsomes catalyze the reduction of ascorbate radical by NADH. The enzyme which functions in this reaction is identified as the microsomal NADH-cytochrome c reductase, the electron transfer system involving cytochrome b₅. Reduced cytochrome b₅, whether bound to or isolated from the microsomal membrane, reacts rapidly with the ascorbate radical to form ascorbate. With intact microsomes, the cytochrome is maintained in the fully reduced state in the presence of NADH. This condition favors the electron transfer from NADH to ascorbate radical as catalyzed by microsomes.
2) The non-ionic detergent, Emalgen 810, which inhibits the reduction of microsomal bound cytochrome b₅ by NADH, also inhibits the NADH-ascorbate radical reductase activity of microsomes.
3) Cytochrome b₅ is reduced by ascorbate very slowly but not reduced by ascorbate radical. Under aerobic condition, the cytochrome appears to be only partially reduced by ascorbate due to the autoxidation of the reduced cytochrome.
4) Microsomes with NADH generating system prevent the oxidation of ascorbate. This effect is inhibited by Emalgen 810, the inhibitor of NADH-cytochrome b₅ reductase system of microsomes. Role of NADH-cytochrome b₅ reductase system to maintain intracellular level of ascorbate is discussed.