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  • Title: Electron paramagnetic resonance study on crosslinked asymmetric Fe(II)-Co(II) hybrids of hemoglobin.
    Author: Kitagishi K, D'Ambrosio C, Yonetani T.
    Journal: Arch Biochem Biophys; 1988 Jul; 264(1):176-83. PubMed ID: 2840023.
    Abstract:
    Asymmetric Fe-Co hybrid hemoglobins (Hbs) containing one, two, and three Co(II) protoporphyrins IX per tetramer have been synthesized. These asymmetric Fe-Co hybrids have been prepared by appropriate combinations of subunits of HbA, HbS, Co(II)HbA, Co(II)HbS, and Fe-Co hybrid Hbs and crosslinking with bis(3,5-dibromosalicyl)fumarate, followed by purification with molecular sieve and ion-exchange chromatography. Electron paramagnetic resonance (EPR) spectra have been measured for the crosslinked asymmetric Fe-Co hybrids to investigate the intermediate ligation states of Hb. The electronic state of the Co(II) ion provides a useful probe to estimate how ligation to iron-containing subunits changes the structure of the cobalt-substituted heme vicinity as described previously (T. Inubushi and T. Yonetani (1983) Biochemistry 22, 1894-1900). The crosslink does not affect the EPR spectra of Fe-Co hybrids. EPR spectra of the alpha (Co) subunits in crosslinked hybrids in the fully deoxy state exhibit a mixture of a broad EPR component and a narrow EPR component which correspond to distorted and undistorted Co(II) coordinate states, respectively, and the ratio of the components varies with pH. EPR spectra of the di-liganded asymmetric hybrid, [alpha (Co) beta (Co)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, and tri-liganded hybrid, [alpha (Co) beta (Fe-CO)]A-[alpha (Fe-CO) beta (Fe-CO)]sXL, show that the coordination state of the alpha (Co) ion in these hybrids is changed to the completely undistorted state in the pH range between 6.5 and 8.8 upon ligation of carbon monoxide (CO). Mono-liganded hybrids, [alpha (Co) beta (Fe-CO)]A-[alpha (Co) beta (Co)]sXL and [alpha (Fe-CO) beta (Co)]A-[alpha (Co) beta (Co)]sXL show intermediate EPR spectra at pH 6.5-7.6, which represent a mixture of the broad and narrow EPR components. The broad component is converted into the narrow one more extensively upon the ligation to the beta (Fe) subunit than to the alpha (Fe) subunit. However, the coordination state of an alpha (Co) subunit in mono-liganded hybrids corresponds to a completely undistorted coordination structure at pH 8.2-8.8. The EPR signal of the alpha (Co) ion is sensitive to the release of alkaline Bohr proton(s) and consequently to the allosteric quaternary structural change of Hb.
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