Structural information through NMR hyperfine shifts in blue copper proteins

The oxidized blue copper proteins azurin and stellacyanin have been investi gated through H-1 NMR at 800 MHz and the results compared with those for pl astocyanin (Bertini, I.; Ciurli, S.; Dikiy, A.; Gasanov, R.; Luchinat, C.; Martini, G.; Safarov, N. J. Am. Chem. Sec. 1999, 121, 2037). By exploiting saturation transfer between the oxidized and the reduced forms, all the hyp erfine shifted signals can be assigned, including the beta-CH2 protons of t he coordinated cysteines, which are so broad not to be detected under direc t observation. Both hyperfine shifts and line widths of the latter signals differ dramatically from one protein to another: average hyperfine shifts o f about 850, 600, and 400 ppm and average line widths of 1.2, 0.45, and 0.2 5 MHz are observed for azurin, plastocyanin, and stellacyanin, in that orde r. The observation of a nuclear line width of 1.2 MHz is unprecedented in h igh-resolution NMR in solution. These data are interpreted as a measure of the out-of-plane displacement of the copper ion, which increases on passing from azurin to plastocyanin to stellacyanin. The present approach seems ge neral for the investigation of blue copper proteins.