Proximal and Distal Effects on the Coordination Chemistry of Ferric Scapharca Homodimeric Hemoglobin As Revealed by Heme Pocket Mutants

Abstract

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-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal 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 coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination 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 retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…We found a cooperative transition, characterized by a pK of 3.7 ± 0.1 and a cooperativity parameter n = 4 (data not shown), which presumably signals denaturation. This interpretation is supported by the presence of a monomeric low-spin hemichrome at acidic pH in the optical spectra of the oxidized (met) HbI species (77,78). A simple Henderson-Hasselbalch transition associated with a single-site His-69 protonation as in MbCO was absent.…”

Estudo espectroscópico do equilíbrio entre espécies hexacoordenada e pentacoordenada dos monômero d nativo e reconstituído da hemoglobina extracelular gigante de Glossoscolex paulistus em meio alcalino

“…Esta saída mais pronunciada do íon férrico causaria o deslocamento hipsocrômico da banda de transferência de carga do ligante para o metal (TCLM) em relação ao pentacoordenado em meio áci-do 23 . De qualquer maneira, Boffi e colaboradores 64 estabeleceram que a presença da banda de transferência de carga, d (íon férrico) → π (heme), tanto em 602 como em 635 nm, indica a presença de complexos de ferro alto-spin, o que corrobora a presente discussão.…”

The Photostabilization of Hemoglobin in Triton X‐100 Aqueous Solution and Triton X‐100/n‐C5H11OH/H2O Microemulsion

“…With the addition of Triton X‐100, the absorption intensity of 500 and 630 nm decreased slightly, while a new absorption peak at 530 nm appeared, which means the formation of hemichrome when considering Fig. 1a (14–16). With the increase in Triton X‐100 concentration, the decrease in absorption intensity at 630 nm and the increase in absorption intensity at 530 nm becomes more distinct, which means that the concentration of hemichrome increases.…”