UV resonance Raman and NMR spectroscopic studies on the pH dependent metal ion release from pseudoazurin

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.