THE SOLUTION STRUCTURE REFINEMENT OF THE PARAMAGNETIC REDUCED HIGH-POTENTIAL IRON-SULFUR PROTEIN-I FROM ECTOTHIORHODOSPIRA-HALOPHILA BY USING STABLE-ISOTOPE LABELING AND NUCLEAR-RELAXATION

The reduced high-potential iron sulfur protein I from Ectothiorhodospi ra halophila which contains the [4Fe-4S](2+) polymetallic center has b een fully labeled with N-15 and C-13. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lo se. Notwithstanding, the solution structure has been reported [Band. L ., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchina t, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Bioche m. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, so ft-HCCH E. COSY and N-15-H-1 correlated NOESY experiments in order to obtain a set of (3)J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of. paramagnetism. From he teronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery ha s been followed from which the contribution to dipolar relaxation of n uclei due to the interaction with the paramagnetic metal ions (rho(par a)) has been estimated. Finally, the intensities of NOEs have been cor rected for the presence of paramagnetic metal ions and these corrected values together with (3)J values and rho(para) data have been used to obtain a well defined solution structure, The aim is that of obtainin g a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, wh ich is anchored to the protein through the rho(para) constraints. In t otal, 1226 corrected NOESY crosspeaks (of which 945 were found to be m eaningful), 37 one-dimensional NOEs, 39 (3)J(HNH alpha) and 37 (3)J(HN C) (providing 45 phi dihedral angle constraints) 54 (3)J(H alpha H bet a) and 31 (3)J(NH beta) (providing 26 chi(1) dihedral angle constraint s), 4 chi(2) dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho( para) constraints, have been used for structure calculation. Restraine d molecular dynamics simulations have also been performed to provide t he final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigatio n of paramagnetic molecules, as the small magnetic moment of heteronuc lei minimizes the paramagnetic influence of unpaired electrons.