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 [4F e-4S] cluster of Desulfovibrio gigas ferredoxin I (FdI). Several low-f ield contact shifted resonances (fast relaxing) are assigned to beta-C H2 and alpha-CH coordinated cysteinyl residues. NOESY patterns (suppor ted by 1D NOE experiments) resolves four pairs of geminal beta-CH2 pro tons at low-field. The cluster ligands are assigned non-specifically t o 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 prove s the occupancy of the fourth site of the [3Fe-4S] complex and implies the coordination of the Cys11 at the cluster. Analysis of the oxidize d form of FdII, using the same methodology as described for FdI, suppo rts the presence of three cysteinyl ligands in the [3Fe-4S] core. Furt her, the combined use of the X-ray coordinates enables the specific as signment of the three cysteinyl ligands of the cluster, extending a pr evious assignment of Cys50. In addition, very broad resonances were de tected for the reduced form of FdII in the low-field region around 200 ppm and in the high field region around -80 ppm.