Abstract:
:UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Stola M,Musiani F,Mangani S,Turano P,Safarov N,Zambelli B,Ciurli Sdoi
10.1021/bi0601003subject
Has Abstractpub_date
2006-05-23 00:00:00pages
6495-509issue
20eissn
0006-2960issn
1520-4995journal_volume
45pub_type
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