High- and low-spin EPR forms of the ferric derivatives of earthworm erythrocruorin

Abstract

THe X-band EPR spectra of both the high and low molecular weight derivatives of earthworm erythrocruorin oxidized with potassium hexacyanoferrate (III) have been investigated. The high molecular weight derivative ( M r 3 · 10 6), obtained by oxidation at pH 7.0, is a typical acid ferric form characterized by an axial type of high-spin signal with g ⊥ near 6. The low molecular weight derivative ( M r 5 · 10 4), obtained by oxidation at pH values different from 7.0, has the absorbance spectrum of a hemichrome. Its EPR spectrum displays several high-spin and low-spin forms that are in pH-dependent equilibrium in the range 6 to 10 and can be assigned to two different heme environments coexisting in the same molecule. This assignment appears clearly from the analysis of the low-spin signals at neutral pH. One low-spin form ( g 1 2.95, g 2 2.28, g 3 1.47) has two histidines protonated at N-1 as apical ligands and is in equilibrium with a low-pH axial high-spin form (p K 7.1) (berne A); the other low-spin form has only one broad temperature-dependent line at approx. g 1 3.35, which can be ascribed to an E-amino:heme:imidazole structure (heme B). As the pH is raised, the bisimidazole species (heme A) undergoes a transition (p K = 9.4) leading to a high-pH axial high-spin form. This can be assigned to a structure in which the heme group is only loosely attached to the protein in accordance with previous absorbance and CD data. The other low-spin species (heme B) is transformed above pH 8.0 into a rhombic high-spin form characterized by a doublet at g 6.64 and g 5.24; this transition cannot be assigned unambiguously to a specific structural change. The existence of two different heme environments in the erythrocruorin hemichrome correlates with the presence of two different distal heme ligands in the oxygenated cobalt-substituted protein.