Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin

The solution structure of the D13C variant of the thermostable Fe7S8 ferred oxin from Bacillus schlegelii has been determined by H-1-NMR spectroscopy i n its oxidized form. In a variable-temperature NMR study the D13C variant w as as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seve nty-five out of 77 amino acid residues and 81% of all theoretically expecte d proton resonances in the D13C Fe8S8 protein have been assigned. Its struc ture was determined through torsion angle dynamics calculations with the pr ogram DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bon d constraints, and NMR-derived dihedral angle constraints fur the cluster-l igating cysteines. Afterwards, restrained energy minimization and restraine d molecular dynamics were applied to each conformer of the family. The fina l family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except For the immediate vic inity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an inse rtion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformation s involving almost half of the amino acid residues was found. The two confo rmations are structurally indistinguishable. Temperature-dependent NMR expe riments show that one of them is thermodynamically more stable than the oth er.