期刊
CHEMISTRY-A EUROPEAN JOURNAL
卷 12, 期 25, 页码 6647-6659出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.200600036
关键词
copper; metalloproteins; nitrite reductase; NMR spectroscopy
Copper-containing nitrite reductases (NiRs) possess type 1 (T1) and type 2 (T2) copper sites and can be either green or blue in color owing to differences at their T1 centers. The active sites of a green and a blue NiR were studied by utilizing their T1Cu(1)/T2Co(II) and T1Co(II)/T2CoII-substituted forms. The UV/Vis spectra of these derivatives highlight the similarity of the T2 centers in these enzymes and that T1 site differences are also present in the Co-II forms. The paramagnetic NMR spectra of T1Cu(I)/T2CoII enzymes allow hyperfine shifted resonances from the three T2 His ligands to be as-signed: these exhibit remarkably similar positions in the spectra of both NiRs, emphasizing the homology of the T2 centers. The addition of nitrite results in subtle alterations in the paramagnetic NMR spectra of the T1Cu(I)/T2Co(II) forms at pH < 7, which indicate a geometry change upon the binding of substrate. Shifted resonances from all of the T1 site ligands have been assigned and the Co-II-N(His) interactions are alike, whereas the (CH)-H-beta proton resonances of the Cys ligand exhibit subtle chemical shift differences in the blue and green NiRs. The strength of the axial Co-II-S(Met) interaction is similar in the two NiRs studied, but the altered conformation of the side chain of this ligand results in a dramatically different chemical shift pattern for the (CH)-H-gamma protons. This indicates an alteration in the bonding of the axial ligand in these derivatives, which could be influential in the Cu-II proteins.
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