H-1-NMR INVESTIGATION OF THE ELECTRONIC-STRUCTURE OF THE 4-IRON FERREDOXIN FROM THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS-LITORALIS

The oxidized and reduced forms of the four-iron ferredoxin from the hy perthermophilic archaeon, Thermococcus litoralis (Tl), have been inves tigated by H-1 NMR spectroscopy. All three protons for each of the fou r Cys are located by 2D and 1D NMR experiments, although differentiati on of alpha- and beta-protons required the interpretation of steady-st ate NOEs and relaxation properties of the signals. The complete correl ation between the Cys signals in the reduced and oxidized ferredoxin w as carried out by 2D EXSY and steady-state saturation-transfer NMR, an d an electron self-exchange rate of similar to 5 x 10(4) M(-1) s(-1) a t 30 degrees C, pH 7.6, is estimated. The beta-protons for two ligated Cys exhibit Curie-like temperature dependence, and the other two disp lay anti-Curie temperature dependence, indicative of the iron atoms ar ising from the mixed-valence pair with intermediate S = 9/2, and the d iferrous pair with intermediate S = 4, respectively. Standard 2D ident ification of the protons for nonligated residues which have significan t dipolar contacts with the ligated Cys provide the sequence-specific assignment for Cys 18 and Cys 16. The two remaining ligated Cys are sh own to be sequence-specifically assignable upon qualitative considerat ion of the relative relaxation properties of the ligated Cys alpha-pro tons, as predicted by the pattern of Cys orientations relative to the cluster in numerous crystallographically characterized bacterial ferre doxins. The combined assignments identify Cys 10 and Cys 16 as ligated to the valence-delocalized iron atoms in the reduced ferredoxin. Comp arison of the NMR spectra of the reduced four-iron and oxidized three- iron Tl ferredoxins indicates a similar pairing of the iron atoms in t he spin coupling hierarchy. The patterns of both relaxation properties and contact shifts of alpha-protons for ligated Cys are discussed in terms of the geometry of the ligand.