PREPARATION AND SPECTROSCOPIC CHARACTERIZATION OF A COUPLED BINUCLEAR CENTER IN COBALT(II)-SUBSTITUTED HEMOCYANIN

被引:51
作者
BUBACCO, L
MAGLIOZZO, RS
BELTRAMINI, M
SALVATO, B
PEISACH, J
机构
[1] YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT MOLEC PHARMACOL,BRONX,NY 10461
[2] UNIV PADUA,DEPT BIOL,I-35131 PADUA,ITALY
[3] UNIV PADUA,CTR BIOCHEM & PHYSIOL HEMOCYANINS & OTHER METALLOPROT,I-35131 PADUA,ITALY
关键词
D O I
10.1021/bi00153a024
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A binuclear cobalt derivative of arthropod hemocyanin (Hc) 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 of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g is similar to 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 is similar to 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due 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 signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the 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 Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.
引用
收藏
页码:9294 / 9303
页数:10
相关论文
共 46 条
[41]  
TAMBURRO AM, 1977, COMP BIOCH PHYSL B, V55, P346
[42]   PHYSICAL STUDIES OF HEMOCYANINS .3. CIRCULAR DICHROISM AND ABSORPTION SPECTRA [J].
VANHOLDE, KE .
BIOCHEMISTRY, 1967, 6 (01) :93-&
[43]   PSEUDO 2-FOLD SYMMETRY IN THE COPPER-BINDING DOMAIN OF ARTHROPODAN HEMOCYANINS - POSSIBLE IMPLICATIONS FOR THE EVOLUTION OF OXYGEN-TRANSPORT PROTEINS [J].
VOLBEDA, A ;
HOL, WGJ .
JOURNAL OF MOLECULAR BIOLOGY, 1989, 206 (03) :531-546
[44]  
WATTERS KL, 1974, INORG CHEM, V13, P752, DOI 10.1021/ic50133a053
[45]  
WEAST RC, HDB CHEM PHYSICS, pB90
[46]   EXAFS STUDIES OF BINUCLEAR COPPER SITE OF OXYZIDOHEMOCYANIN, DEOXYZIDOHEMOCYANIN, METAQUOZIDOHEMOCYANIN, METFLUOROZIDOHEMOCYANIN, AND METAZIDOHEMOCYANIN FROM ARTHROPODS AND MOLLUSKS [J].
WOOLERY, GL ;
POWERS, L ;
WINKLER, M ;
SOLOMON, EI ;
SPIRO, TG .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1984, 106 (01) :86-92