EXTRACELLULAR FERRIREDUCTASE ACTIVITY OF K562 CELLS IS COUPLED TO TRANSFERRIN-INDEPENDENT IRON TRANSPORT

The reduction of Fe3+ to Fe2+ has been established to play a critical role in the uptake of iron by many organisms. Recently, a mechanism of iron transport in the absence of transferrin (Tf) was described for the human K562 cell line and a role for ferrireductase activity was implicated in this process as well [Inman, R. S., & Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528]. The present report characterizes the extracellular reduction of ferricyanide to ferrocyanide catalyzed by K562 cells. The observation that membrane-impermeant ferricyanide competitively inhibits Tf-independent assimilation of iron from Fe-55-nitriloacetic acid indicates that this ferrireductase activity is indeed coupled to the transport mechanism. From a series of initial rate experiments, the kinetic parameters for cell surface ferrireductase activity, V-max = 0.102 nmol min(-1) (10(6) cells)(-1) and K-m = 6.13 mu M, were determined. Neither the V-max nor the K-m of this reaction is modulated by changes in extra- or intracellular iron levels; thus, similar to Tf-independent transport activity in K562 cells, the ferrireductase activity is not regulated in response to iron levels. Transmembrane oxidoreductase activity is also reportedly involved in the control of cellular growth; however, the K562 cell ferrireductase is unresponsive to insulin and is not inhibited by the antitumor drugs adriamycin, actinomycin D, or cis-platin, observations that fail to support a role for this particular activity in cell regulation. Rather, the K562 cell ferrireductase appears to be tightly coupled to the mechanism of Tf-independent transport as demonstrated by its sensitivity to Cd2+, a specific inhibitor of non-Tf iron uptake by K562 cells. Cd2+ blocks the reduction of ferricyanide catalyzed by K562 cells with an IC50 similar to 35 mu M, a value close to that determined for inhibition of iron transport. Moreover, Cd2+ inhibits transport in the presence of ascorbate, a chemical reducing agent, suggesting that the substrate binding site for reduction and transport are one in the same and that both of these functions may be carried out by the same molecular complex.