Elucidation of the Paramagnetic R1 Relaxation of Heteronuclei and Protons in Cu(II) Plastocyanin from Anabaena variabilis

Abstract

The longitudinal paramagnetic dipolar relaxation rates, R1p, of 15N, 13C, and 1H nuclei in plastocyanin from Anabaena variabilis (A.v. PCu) were determined at 11.7 and 17.6 T from the corresponding experimental relaxation rates in reduced (R1d) and partly oxidized (R1o) A.v. PCu. To obtain an accuracy of the relaxation data sufficiently high for the subsequent analysis, the experimental rates were determined by a simultaneous least-squares analysis of all the spectra in a relaxation experiment. Also, a refined solution structure of A.v. PCu was determined from 1459 NOE distance restraints and 87 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The average rms deviation from the mean structure of the 20 structures with the lowest total energy is 0.75 Å for the backbone atoms and 1.21 Å for all heavy atoms. The distance information of the dipolar paramagnetic R1p rates was compared with the corresponding distances in the refined NMR solution structure. The comparison reveals that the point dipolar approximation, which assumes that R1p is caused by a dipolar interaction of the nuclei with the metal-centered unpaired electron of the Cu2+ ion, does not apply to the heteronuclei. In the case of protons it applies only for proton−copper distances shorter than ∼10 Å. In contrast, it is found that the R1p relaxation of the 15N and 13C nuclei is dominated by dipolar interaction with unpaired metal electron spin density delocalized onto the 2pz orbitals of the heteronuclei. From the R1p rates of the heteronuclei and the metal−nuclei distances in the refined NMR solution structure, the delocalized unpaired spin densities ρπ of the individual 15N and 13C nuclei were derived. It is found that ρπ decays approximately exponentially with the metal−nuclei distance and almost isotropically throughout the protein. Possible implications of this decay for the electron-transfer pathways of A.v. PCu are discussed.