Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coo rdinate derivative, and a dimeric high-spin five-coordinate species that pr evail at acidic, neutral, and alkaline pH values, respectively. In the five -coordinate derivative, the iron atom is bound to a hydroxyl group on the d istal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordi nation and spin state of the iron atom and on the association state, two he me pocket mutants have been investigated by means of optical absorption, re sonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the co ordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is reta ined, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate de rivative at neutral and alkaline pH values. At variance with native HbI, th e His69 --> Val mutant is always high-spin and does not undergo dissociatio n into monomers at acidic pH values. The Phe97 --> Leu mutant, like the nat ive protein, forms a monomeric hemichrome species at acidic pH values. Howe ver, at alkaline pH, it does not give rise to the unusual hydroxyl-bound fi ve-coordinate derivative but forms a six-coordinate derivative with the pro ximal His and distal hydroxyl as iron ligands.