Most of [4Fe-4S] proteins bind their metallic center by four cysteine residues, three clustered in a single stretch of seven amino acids and a remote fourth generally followed by a proline residue. Two such prolines in Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been substituted by different amino acids and the resulting molecular variants studied with EPR and NMR spectroscopies. The isolated EPR contributions of the [4Fe-4S]+ clusters do not change much in all variants. The exact positions or the number of features composing the fully reduced EPR spectra built by the two interacting [4Fe-4S]+ S = 1/2 systems vary slightly but, in none of the proteins in which either proline 19 or 48 were substituted, do they indicate a major difference either in the folding of the ferredoxin or in the electronic structure of its clusters. A subset of paramagnetically shifted NMR signals is significantly affected by these replacements at both redox levels. The corresponding protons belong to two cysteines liganding the cluster close to the substitution. These data, combined with the presently available three-dimensional information, form the basis for partial assignments of the most shifted resonances in the NMR spectra of such proteins. The positions of intermediate lines in the NMR spectra of semireduced ferredoxins depend on the difference between the redox potentials of the two clusters; this difference is sensitive to the substitutions of either conserved proline residue by lysine.