2-DIMENSIONAL PULSED EPR SPECTROSCOPY OF THE COPPER PROTEIN AZURIN

Two-dimensional (2D) pulsed EPR spectroscopy was applied to study the copper ligands in azurins from Pseudomonas aeruginosa, Az(pae), and Al caligenes species NCIIB 11015, Az(asp), in frozen solutions. While a h igh-resolution three-dimensional crystal structure is available for Az (pae), only a low-resolution structure has been reported for Az(asp). Az(pae) was studied in the pH range 3.9-7.0 and Az(asp) at a pH of 4.8 . Measurements were performed at 9 GHz which is usually within the can cellation condition for the remote nitrogen of imidazole ligands. The main technique was the hyperfine sublevel correlation (HYSCORE) techni que. At all pH values investigated the 2D HYSCORE spectra of Az(pae) s howed correlations between the nuclear frequencies corresponding to th e nuclear quadrupole resonance (NQR) frequencies of the remote nitroge ns of the imidazole ligands and the double quantum frequency. The spec tra showed additional well-resolved cross peaks which indicate correla tions between the NQR frequencies of a weakly coupled amide nitrogen a nd the corresponding double quantum frequency. This confirms earlier d etection and assignment of the electron spin-echo envelope modulation (ESEEM) frequencies of this nitrogen which were based on ESEEM measure ments of the H117G mutant (Coremans et al. Chem. Phys. Lett. 1995, 235 , 202). The 2D spectra of Az(asp) were similar to those of Az(pae) sho wing that a third weakly coupled nitrogen is present in this species a s well. HYSCORE spectra of a frozen solution of ascorbate oxidase exhi bited only signals corresponding to the remote nitrogens of the imidaz ole. Comparing these spectra with those of the azurins and correlating the results with the available crystal structures of ascorbate oxidas e and Az(pae) suggest that the third nitrogen in Az(pae) is the amide nitrogen of His-46, coupled to the copper via the carbonyl group of Gl y-45. This further implies that also in azurin from Az(asp), the preci se 3D structure of which is not yet available, the copper has five lig ands rather than four, This study demonstrates that the 2D HYSCORE exp eriment is most useful for detecting, unraveling, and assigning ESEEM frequencies in metalloproteins.