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 Scapharca inaequivalvis is characterized by unusual properties of the ferric derivative. The dimeric aquomet form undergoes' a pH-dependent reversible 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 reduced HbI-CO adduct is obtained readily in the presence of carbon monoxide. The ferrocyanide binding site of HbI has been identified by modifying 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 oxidation by ferricyanide depends on the presence and position of a negatively charged group on the aromatic ring, indicating that the binding site 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 reaction between bound ferrocyanide and the heme iron confirmed this location. The proposed binding site of the ferrocyanide anion comprises a cluster of positive charges at the subunit interface formed by Lys 96, Arg 53', Lys 65', and Arg 67' where apices indicate residues of the contralateral subunit,