STRUCTURAL CHARACTERIZATION OF MONONUCLEAR CU(II) AND ITS NITRITE COMPLEX IN THE ACTIVE-SITE OF CARCINUS-MAENAS HEMOCYANIN

被引:15
作者
BUBACCO, L
MAGLIOZZO, RS
WIRT, MD
BELTRAMINI, M
SALVATO, B
PEISACH, J
机构
[1] YESHIVA UNIV ALBERT EINSTEIN COLL MED,DEPT MOLEC PHARMACOL,BRONX,NY 10461
[2] UNIV PADUA,CTR BIOCHEM & PHYSIOL HEMOCYANINS & OTHER MET PR,I-35131 PADUA,ITALY
关键词
D O I
10.1021/bi00005a008
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
The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three Ligands at 1.99 +/- 0.01 Angstrom and a fourth one at 2.26 +/- 0.01 Angstrom from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESEEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water Ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and A(Cu) values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.
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收藏
页码:1524 / 1533
页数:10
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