SOLUTION STRUCTURE OF THE OXIDIZED 2[4FE-4S] FERREDOXIN FROM CLOSTRIDIUM-PASTEURIANUM

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimension al structure in solution of the oxidized Clostridium pasteurianum ferr edoxin has been solved by H-1-NMR. The X-ray structure is not availabl e. The protein contains 55 amino acids and two [4Fe-4S] clusters. In t he oxidized state, the clusters have S = 0 ground states, but are para magnetic because of thermal population of excited states. Due to the s omewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non -selective T-1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology bot h in achieving an extended assignment and in obtaining a suitable numb er of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar conne ctivities, partially detected through tailored experiments, (3)J(HN-H alpha), H-bond constraints and dihedral angle constraints on the Cys c hi(2) angles have been generated by using a recently derived Karplus-t ype relationship for the hyperfine shifts of cysteine beta CH2 protons . In total, 456 constraints have been used in distance geometry calcul ations. The final quality of the structures is satisfactory, with root -mean-square deviation values of 66 pm and 108 pm for backbone and hea vy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duee, E. D., Fanchon, E., Vicat , J., Sieker, L. C., Meyer, J. and Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall foldin g, secondary structure elements and side-chain orientations. The C alp ha root-mean-square deviation values between the X-ray-determined C, a cidi urici ferredoxin structure and the conformer with lowest energy o f the C, pasteurianum ferredoxin family is 78 pm (residues 3-53). Disc repancies in residues 26-28 may arise from the disorder observed in th e X-ray structure in that region.