Selective observation of the Cu(I)-amicyanin metal site by paramagnetic NMR on partially oxidised samples.

The relaxation enhancement caused by paramagnetic copper(II) is used to observe selectivelythe metal site of copper(I)-amicyanin by one- and two-dimensional NMR spectroscopy. Theparamagnetic effect is communicated to the diamagnetic protein through the electron self-exchange reaction in partially oxidised samples, and can be used for the selective detectionof protons around the metal. Relaxation-selective NMR pulse sequences, like super-WEFTand WEFT-NOESY, are used to achieve the desired selection of the signals. The spectraobtained show well-resolved signals corresponding to protons within a radius of approximately 7 A from the metal, including almost all protons from the coordinated residues. A significant increasein resolution as well as selection of the most relevant part of the protein (close to the activecentre) are the principal advantages of this technique, which can be used to obtain specificinformation about the metal site in blue copper proteins, to assist in the assignment of theirNMR spectra and to determine functional properties like the electron self-exchange rate.