Elucidation of the paramagnetic R-1 relaxation of heteronuclei and protonsin Cu(II) plastocyanin from Anabaena variabilis

The longitudinal paramagnetic dipolar relaxation rates, R-1p, of N-15, C-13 , and H-1 nuclei in plastocyanin from Anabaena variabilis (A. v. PCu) were determined at 11.7 and 17.6 T from the corresponding experimental relaxatio n rates in reduced (R-1d) and partly oxidized (R-1o) A. v. PCu. To obtain a n accuracy of the relaxation data sufficiently high for the subsequent anal ysis, the experimental rates were determined by a simultaneous least-square s analysis of all the spectra in a relaxation experiment. Also, a refined s olution structure of A. v. PCu was determined from 1459 NOE distance restra ints and 87 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The average rms deviation from the mean st ructure of the 20 structures with the lowest total energy is 0.75 Angstrom for the backbone atoms and 1.21 Angstrom for all heavy atoms. The distance information of the dipolar paramagnetic R-1p rates was compared with the co rresponding distances in the refined NMR solution structure. The comparison reveals that the point dipolar approximation, which assumes that R-1p is c aused by a dipolar interaction of the nuclei with the metal-centered unpair ed electron of the Cu2+ ion, does not apply to the heteronuclei. In the cas e of protons it applies only for proton-copper distances shorter than simil ar to 10 Angstrom. In contrast, it is found that the R-1p relaxation of the N-15 and C-13 nuclei is dominated by dipolar interaction with unpaired met al electron spin density delocalized onto the 2p(z) orbitals of the heteron uclei. From the R-1p rates of the heteronuclei and the metal-nuclei distanc es in the refined NMR solution structure, the delocalized unpaired spin den sities rho(pi) of the individual N-15 and C-13 nuclei were derived. It is f ound that rho(pi) decays approximately exponentially with the metal-nuclei distance and almost isotropically throughout the protein. Possible implicat ions of this decay for the electron-transfer pathways of A. v. PCu are disc ussed.