Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A H-1 NMR study

被引:47
作者
Salgado, J
Jimenez, HR
Moratal, JM
Kroes, S
Warmerdam, GCM
Canters, GW
机构
[1] UNIV VALENCIA, DEPT INORGAN CHEM, E-46100 BURJASSOT, VALENCIA, SPAIN
[2] LEIDEN UNIV, GORLAEUS LABS, 2300 RA LEIDEN, NETHERLANDS
关键词
D O I
10.1021/bi951748a
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
Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic H-1 NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin. Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gin in stellacyanin.
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页码:1810 / 1819
页数:10
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