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

Abstract

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