2-DIMENSIONAL H-1-NMR STUDIES ON DESULFOVIBRIO-GIGAS FERREDOXINS - ASSIGNMENT OF THE IRON-SULFUR CLUSTER CYSTEINYL LIGAND PROTONS

1D and 2D H-1 NMR studies are reported on the oxidized and reduced [4Fe-4S] cluster of Desulfovibrio gigas ferredoxin I (FdI). Several low-field contact shifted resonances (fast relaxing) are assigned to beta-CH2 and alpha-CH coordinated cysteinyl residues. NOESY patterns (supported by 1D NOE experiments) resolves four pairs of geminal beta-CH2 protons at low-field. The cluster ligands are assigned non-specifically to Cys8, Cys11, Cys14 and Cys50, based on the X-ray structural analysis available for the oligomeric form, FdII, that contains a single [3Fe-4S] cluster. It was indicated in this case that Cys11 is not bound to the trinuclear cluster but is tilted towards the solvent. The presence of four pairs of geminal beta-CH2 protons for FdI unambiguously proves the occupancy of the fourth site of the [3Fe-4S] complex and implies the coordination of the Cys11 at the cluster. Analysis of the oxidized form of FdII, using the same methodology as described for FdI, supports the presence of three cysteinyl ligands in the [3Fe-4S] core. Further, the combined use of the X-ray coordinates enables the specific assignment of the three cysteinyl ligands of the cluster, extending a previous assignment of Cys50. In addition, very broad resonances were detected for the reduced form of FdII in the low-field region around 200 ppm and in the high field region around -80 ppm.