Electron Transfer Reactions of Candidate Tumor Suppressor 101F6 Protein, a Cytochrome b₅₆₁ Homologue, with Ascorbate and Monodehydroascorbate Radical

The candidate tumor suppressor 101F6 protein is a homologue of adrenal chromaffin granule cytochrome b₅₆₁, which is involved in the electron transfer from cytosolic ascorbate to intravesicular monodehydroascorbate radical. Since the tumor suppressor activity of 101F6 was enhanced in the presence of ascorbate, it was suggested that 101F6 might utilize a similar transmembrane electron transfer reaction. Detailed kinetic analyses were conducted on the detergent-solubilized recombinant human 101F6 for its electron transfer reactions with ascorbate and monodehydroascorbate radical by stopped-flow and pulse radiolysis techniques. The reduction of oxidized 101F6 with ascorbate was found to be independent of pH in contrast to those observed for chromaffin granule and Zea mays cytochromes b₅₆₁ in which both cytochromes exhibited very slow rates at pH 5.0 but faster at pH 6.0 and 7.0. The absence of the inhibition for the electron acceptance from ascorbate upon the treatment with diethyl pyrocarbonate suggested that 101F6 might not utilize a “concerted proton/electron transfer mechanism”. The second-order rate constant for the electron donation from the ascorbate-reduced 101F6 to the pulse-generated monodehydroascorbate radical was found to be 5.0 × 10⁷ M^–1 s^–1, about 2-fold faster than that of bovine chromaffin granule cytochrome b₅₆₁ and about five times faster than that of Zea mays cytochrome b₅₆₁, suggesting that human 101F6 is very effective for regenerating ascorbate from monodehydroascorbate radical in cells. Present observations suggest that 101F6 employs distinct electron transfer mechanisms on both sides of the membranes from those of other members of cytochrome b₅₆₁ protein family.