The Nickel Site of Bacillus pasteurii UreE, a Urease Metallo-Chaperone, As Revealed by Metal-Binding Studies and X-ray Absorption Spectroscopy
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- 作者：Massimiliano Stola ; Francesco Musiani ; Stefano Mangani ; Paola Turano ; Niyaz Safarov ; Barbara Zambelli ; and Stefano Ciurli
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：May 23, 2006
- 年：2006
- 卷：45
- 期：20
- 页码：6495 - 6509
- 全文大小：384K
- 年卷期：v.45,no.20(May 23, 2006)
- ISSN：1520-4995
文摘
UreE is a homodimeric metallo-chaperone that assists the insertion of Ni2+ ions in the activesite of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have beendetermined, but the details of the nickel-binding site were not elucidated due to solid-state effects thatcaused disorder in a key portion of the protein. A complementary approach to this problem is describedhere. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni2+, followed by metal ion quantitativeanalysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the bindingof 2 Ni2+ ions to the functional dimer, with an overall dissociation constant KD = 35 
M. To establishthe nature, the number, and the geometry of the ligands around the Ni2+ ions in BpUreE-Ni2, X-rayabsorption spectroscopy data were collected and analyzed using an approach that combines ab initioextended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possiblecoordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni2+ ionsin octahedral coordination geometry and an average of two histidine residues and four O/N ligands boundto each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Nimoiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. Theseresults were interpreted using two possible models. One model involves the presence of two identicalmetal sites binding Ni2+ with negative cooperativity, with each metal ion bound to the conserved His100as well as to either His145 or His147 from each monomer, residues found largely conserved at the C-terminal.The alternative model comprises the presence of two different binding sites featuring different affinityfor Ni2+. This latter model would involve the presence of a dinuclear metallic core, with one Ni2+ ionbound to one His100 from each monomer, and the second Ni2+ ion bound to a pair of either His145 orHis147. The arguments in favor of one model as compared to the other are discussed on the basis of theavailable biochemical data.
M. To establishthe nature, the number, and the geometry of the ligands around the Ni2+ ions in BpUreE-Ni2, X-rayabsorption spectroscopy data were collected and analyzed using an approach that combines ab initioextended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possiblecoordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni2+ ionsin octahedral coordination geometry and an average of two histidine residues and four O/N ligands boundto each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Nimoiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. Theseresults were interpreted using two possible models. One model involves the presence of two identicalmetal sites binding Ni2+ with negative cooperativity, with each metal ion bound to the conserved His100as well as to either His145 or His147 from each monomer, residues found largely conserved at the C-terminal.The alternative model comprises the presence of two different binding sites featuring different affinityfor Ni2+. This latter model would involve the presence of a dinuclear metallic core, with one Ni2+ ionbound to one His100 from each monomer, and the second Ni2+ ion bound to a pair of either His145 orHis147. The arguments in favor of one model as compared to the other are discussed on the basis of theavailable biochemical data.