IDENTIFICATION OF THE SITE OF FERROCYANIDE BINDING INVOLVED IN THE INTRAMOLECULAR ELECTRON-TRANSFER PROCESS TO OXIDIZED HEME IN SCAPHARCA DIMERIC HEMOGLOBIN

The cooperative homodimeric hemoglobin (HbI)(2) from the mollusc Scaph arca inaequivalvis is characterized by unusual properties of the ferri c derivative. The dimeric aquomet form undergoes' a pH-dependent rever sible dissociation into a monomeric low-spin hemichrome. Moreover, in HbI oxidized with ferricyanide the ferrocyanide anion produced in the reaction remains bound to the oxidized protein with high affinity and forms an intramolecular redox couple with the heme iron. Thus, the red uced HbI-CO adduct is obtained readily in the presence of carbon monox ide. The ferrocyanide binding site of HbI has been identified by modif ying the only cysteine residue of the polypeptide chain, Cys 92 (F2), which is located at the subunit interface near the proximal histidine (His 101, F11). In HbI modified with organomercurials the rate of oxid ation by ferricyanide depends on the presence and position of a negati vely charged group on the aromatic ring, indicating that the binding s ite of the ferrocyanide anion is located near Cys 92. The tendency to dissociate into the monomeric hemichrome of the various Cys ga-reacted proteins and the study of the intramolecular electron transfer reacti on between bound ferrocyanide and the heme iron confirmed this locatio n. The proposed binding site of the ferrocyanide anion comprises a clu ster of positive charges at the subunit interface formed by Lys 96, Ar g 53', Lys 65', and Arg 67' where apices indicate residues of the cont ralateral subunit, (C) 1994 Academic Press, Inc.