Open AccessSolution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum
Author(s) -
Bertini Ivano,
Donaire Antonio,
Feinberg Benjamin A.,
Luchinat Claudio,
Picciolp Mario,
Yuan Huaiping
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.tb20799.x
Subject(s) - ferredoxin , chemistry , dihedral angle , paramagnetism , crystallography , proton , excited state , protein structure , population , cysteine , molecule , physics , atomic physics , hydrogen bond , quantum mechanics , organic chemistry , biochemistry , demography , sociology , enzyme
Following the recently developed approach to the solution structure of paramagnetic high‐potential iron‐sulfur proteins, the three‐dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1 H‐NMR. The X‐ray structure is not available. The protein contains 55 amino acids and two [4Fe‐4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat 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 both in achieving an extended assignment and in obtaining a suitable number 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 connectivities, partially detected through tailored experiments, 3 J HN ‐ Hα , H‐bond constraints and dihedral angle constraints on the Cys X 2 angles have been generated by using a recently derived Karplus‐type relationship for the hyperfine shifts of cysteine βCH 2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root‐mean‐square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.‐M. (1994) J. Mol Biol. 243 , 683–695]. The two proteins are very similar in the overall folding, secondary structure elements and side‐chain orientations. The Cα root‐mean‐square deviation values between the X‐ray‐determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3–53). Discrepancies in residues 26–28 may arise from the disorder observed in the X‐ray structure in that region.