Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.
Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.
复制标题
钴（II）取代的血蓝蛋白中偶联双核中心的制备和光谱表征。
DOI：
10.1021/bi00153a024
复制
发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Peisach,J
中科院分区：
文献类型：
--
作者：
Bubacco,L;Magliozzo,RS;Beltramini,M;Salvato,B;Peisach,J
Revised Manuscript Received July 9, 1992 abstract: A binuclear cobalt derivative of arthropod hemocyanin (He) has been prepared by the reaction of apo-Hc with Co (II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co (II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients ofCo (II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co (II) derivative contains a resonance at g~ 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co (II) bound to crude products exhibits an EPR signal at g~ 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorptionin the visible regiondue to Co (II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co (II) per protein monomer], but the intensity of the Co (II) EPR signalincreases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h tothe formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co (II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native He withCu (I), indicating thestable loading of Co (II) in the active site. EPR and optical spectroscopic evidence is presentedshowing that the binuclear derivative does not bind oxygen.
