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

Abstract

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 × 10(7) 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.

Elucidation of molecular functions of human tumor suppressor protein 101F6 by reconstitution into phospholipid bilayer nanodiscs

“…Expression and purification of 101F6 protein were conducted by employing alcohol-assimilating yeast Pichia pastoris cells and pPICZB-101F6-His8 plasmid (Recuenco et al 2013a, Recuenco et al 2013b. In brief, human 101F6 gene was incorporated into the Pichia genome and successful transformants were selected by zeocin-resistance screening.…”

Iron metabolism and its contribution to cancer (Review)

“…As a member of the cytochrome b561 family, Dcytb serves a vital role in the ascorbate-dependent reduction of inorganic iron in duodenal enterocytes. Besides Dcytb, there are additional members of the cytochrome b561 family, including stromal cell-derived receptor 2, cytochrome b ascorbate-dependent protein 3 and cytochrome b561 domain-containing protein 2 (33,34). Dcytb reduces extracellular ferric iron to ferrous ion (24,35).…”

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity

“…However, for other members of the cytochrome b 561 protein family, the presence of the ferric resuctase activity was not proved yet on the molecular level. 101F6 protein differs significantly from Cecytb-2 protein (or other classic members of cytochrome b 561 family) in amino acid sequences (belonging to a very distant subfamily) and in their biophysical properties (EPR spectra, redox potentials, reactivity with ascorbate) [24][25][26]. Therefore, the molecular mechanism of the ferric reductase activity of 101F6 may be very different from those of usual cytochromes b 561 .…”