Journal article
Two-dimensional pulsed EPR spectroscopy of the copper protein azurin
Journal of the American Chemical Society, Vol.118(5), pp.1201-1206
Feb/1996
Abstract
Two-dimensional (2D) pulsed EPR spectroscopy was applied to study the copper ligands in azurins from Pseudomonas aeruginosa, Az(pae), and Alcaligenes species NCIIB 11015, Az(asp), in frozen solutions. While a high-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 cancellation condition for the remote nitrogen of imidazole ligands. The main technique was the hyperfine sublevel correlation (HYSCORE) technique. At all pH values investigated the 2D HYSCORE spectra of Az(pae) showed correlations between the nuclear frequencies corresponding to the nuclear quadrupole resonance (NQR) frequencies of the remote nitrogens of the imidazole ligands and the double quantum frequency. The spectra showed additional well-resolved cross peaks which indicate correlations between the NQR frequencies of a weakly coupled amide nitrogen and the corresponding double quantum frequency. This confirms earlier detection and assignment of the electron spin-echo envelope modulation (ESEEM) frequencies of this nitrogen which were based on ESEEM measurements 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) showing that a third weakly coupled nitrogen is present in this species as well. HYSCORE spectra of a frozen solution of ascorbate oxidase exhibited only signals corresponding to the remote nitrogens of the imidazole. Comparing these spectra with those of the azurins and correlating the results with the available crystal structures of ascorbate oxidase 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 Gly-45. This further implies that also in azurin from Az(asp), the precise 3D structure of which is not yet a
Details
- Title
- Two-dimensional pulsed EPR spectroscopy of the copper protein azurin
- Creators
- V Kofman (null)O Farver (null)Israel Pecht (null) - The Weizmann Institute of ScienceDaniella Goldfarb (null) - 972WIS_INST___136
- Resource Type
- Journal article
- Publication Details
- Journal of the American Chemical Society, Vol.118(5), pp.1201-1206; Feb/1996
- Number of pages
- 6
- Language
- English
- DOI
- https://doi.org/10.1021/ja952704s
- Record Identifier
- 993262750803596
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