Lookup NU author(s): Dr Katsuko Sato,
Professor Christopher Dennison
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UV resonance Raman (UVRR) and 1H NMR spectra are measured for native Cu(I)- and Cu(II)-pseudoazurin, its apo-protein, and a few metal-substituted derivatives. The pH titration experiments of 1H NMR enabled us to determine the pKa* values of three His residues (His6, His40, and His81). The UVRR band characteristic of a metal coordinated histidyl imidazole was observed at 1385 cm-1 for Cu(II)-pseudoazurin in D2O but not for Cu(I)-pseudoazurin. This frequency is consistent with the Nδ coordination of His. For the Cu(I)-pseudoazurin a characteristic band of histidyl imidazolium was detected at 1408 cm-1 at acidic pH. This is assigned to protonated His81, which is deligated from Cu(I) at low pH values. The imidazolium Raman band at 1408 cm-1 was also detected in the UVRR spectrum of apo-pseudoazurin at pH* 3.9, and the acidification was accompanied by a significant change in the X-Pro bands at 1467 cm-1. Pseudoazurin substituted with Zn2+ and Cd2+ gave the characteristic Raman bands of the metal coordinated imidazole at 1388 and 1384 cm-1, respectively, at neutral pH, but its intensity diminished upon lowering the pH, and instead the imidazolium band at 1408 cm-1 grew. The X-Pro bands of the pseudoazurins substituted with Zn2+ and Cd2+ exhibited the pH dependence very similar to that observed for apo-pseudoazurin. These findings indicate that the Zn2+ and Cd2+ ions are released from the active site at acidic pH and it is accompanied by a change in hydrogen bonding state of Pro80. This behavior is clearly absent in both the Cu(I) and Cu(II) proteins, meaning that pseudoazurin discriminates between copper and the other metal ions. © 2002 Elsevier Science B.V. All rights reserved.
Author(s): Sato K; Dennison C; Nagatomo S; Niizeki T; Kitagawa T; Kohzuma T
Publication type: Article
Publication status: Published
Journal: Inorganica Chimica Acta
ISSN (print): 0020-1693
ISSN (electronic): 1873-3255
Publisher: Elsevier BV
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