Use of H-1 longitudinal relaxation times in the solution structure of paramagnetic proteins, application to [4Fe-4S] proteins

The accuracy of the solution structures determined by NMR is often poor around paramagnetic centers because the properties of the near protons are strongly perturbed by the electronic spin. The structural information contained in the relaxation rates of these protons has been extracted here by measuring the longitudinal relaxation times with the inversion-recovery total correlation spectroscopy (IR-TOCSY) sequence based on the recovery of cross peaks. In addition to measurements with nonselective inversion-recovery for nonoverlapping signals, reliable data have been obtained for a majority of main-chain protons from Chromatium vinosum high-potential ferredoxin. When a small and constant contribution from diamagnetism as well as the electronic spin distribution over the [4Fe-4S] cluster are taken into account, the shortest longitudinal relaxation times depend directly on the distance separating the protons from the paramagnetic center. This indicates that electron-nuclei dipolar interactions are the most efficient relaxation mechanism for these protons. However, the expected dependence of the relaxation rates as the sixth power of the distance has to be corrected because of induced relaxation among fast relaxing protons. This approach reveals that the solution structure of the protein is significantly different from the crystal structure around Phe-48. In addition, it provides an independent confirmation of the actual electronic structure of the [4Fe-4S](3+) cluster in the protein. The method devised in this work, which does not rely on specific enrichment, should be useful to improve the determination of NMR-derived solution structures of paramagnetic macromolecules.