Sulfheme Proteins: I. OPTICAL AND MAGNETIC PROPERTIES OF SULFMYOGLOBIN AND ITS DERIVATIVES

Abstract

Abstract Ferrous sulfmyoglobin was prepared from the higher oxidation state of myoglobin (Mbiv) by reaction at pH 8 with a 1.5-fold molar excess of (NH4)2S. The purity (g90%) was greater than any previously reported. The electron paramagnetic resonance (EPR) of the ferric protein at low pH resembles that of ferric myoglobin, where the heme iron is high spin and is in a nearly axial environment. At high pH, the heme iron gives a low spin EPR signal, attributed to the hydroxide derivative, which is distinct from that of ferric myoglobin hydroxide. Acid-base titration of ferric sulfmyoglobin in the visible and near infrared gives a pK of 8.45 ± 0.05 while similar titration of ferric myoglobin yields a pK of 8.9 to 9.0. A computer analysis of the titration data for both compounds confirmed the pK for ferric sulfmyoglobin and, coupled with EPR data, has been used to determine the purity of the preparation. Evidence is presented that the small amount of impurity in the ferrous sulfmyoglobin preparation is present as oxymyoglobin. A computed spectrum of pure ferrous sulfmyoglobin is given, and has been used to re-evaluate the purity of preparations described in previous publications. Fluoride binding to ferric sulfmyoglobin was established by EPR. The low spin ferric cyanide and azide derivatives were studied by optical and EPR spectroscopy. An analysis of the EPR spectra of low spin derivatives of ferric sulfmyoglobin leads us to believe that the electronic distribution at the heme is different from that of ferric myoglobin and, combined with an analysis of optical properties, supports the hypothesis of a structure in which the elements of H2S add across a β—β double bond of a pyrrole and thereby disrupt the porphyrin conjugation and form a chlorin type structure.