Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins of the cell. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates metabolic, ion transport and mechanical properties of the cell. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 with high affinity, these observations raise the hypothesis that hemoglobin (Hb) might regulate erythrocyte properties through its oxygenation-dependent association with band 3. To explore how reversible association of deoxyHb with band 3 might modulate erythrocyte properties, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that:
the deoxyHb binding site on the erythrocyte membrane comprises residues 12–23 of band 3;
mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction was possible had it been beneficial;
Hb does not bind to two similar sequences in band 3 despite their high sequence homology to residues 12–23, and
the Hb binding site on band 3 lies proximal to binding sites for glycolytic enzymes, band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by Hb oxygenation.
We conclude that nature has evolved a deoxyHb binding site on band 3 whose affinity is regulated by O2, enabling the reversible association of deoxyHb with band 3 to serve as a regulator of the oxygenation-dependent properties of the cell.
Disclosure: No relevant conflicts of interest to declare.