EFFECT OF REPLACING CONSERVED PROLINE RESIDUES ON THE EPR AND NMR PROPERTIES OF CLOSTRIDIUM-PASTEURIANUM-2[4FE-4S] FERREDOXIN

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 pro lines in Clostridium pasteurianum 2 [4Fe-4S] ferredoxin have been subs tituted by different amino acids and the resulting molecular variants studied with EPR and NMR spectroscopies. The isolated EPR contribution s of the [4Fe-4S]+ clusters do not change much in all variants. The ex act positions or the number of features composing the fully reduced EP R 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 f olding of the ferredoxin or in the electronic structure of its cluster s. A subset of paramagnetically shifted NMR signals is significantly a ffected by these replacements at both redox levels. The corresponding protons belong to two cysteines liganding the cluster close to the sub stitution. These data, combined with the presently available three-dim ensional information, form the basis for partial assignments of the mo st shifted resonances in the NMR spectra of such proteins. The positio ns of intermediate lines in the NMR spectra of semireduced ferredoxins depend on the difference between the redox potentials of the two clus ters; this difference is sensitive to the substitutions of either cons erved proline residue by lysine.