Solution Structure of the Oxidized 2[4Fe‐4S] Ferredoxin from Clostridium Pasteurianum

Abstract

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 'H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two 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 , 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, 3Jm.H0, H-bond constraints and dihedral angle constraints on the Cys x2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine PCH, 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 uriciBiol. 243, 683-6951. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The Ca 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.Keywords: NMR ; solution structure ; paramagnetic metalloproteins ; ferredoxins ; iron-sulfur proteins.It has been recently shown that it is possible to obtain threedimensional structures of paramagnetic proteins in solution [ 1, 21 (Bertini, I., Eltis, D., Felli, I. C., Kastrau, D. H. W., Luchinat, C. and Piccioli, M., unpublished results). This can be achieved by combining classical NMR methods for structure determination [3-51 with tailored experiments to detect connectivities involving fast relaxing signals [6-101. It is known that the feasibility of a solution structure in the case of diamagnetic systems is dependent essentially on the molecular mass of the protein [1 11. In paramagnetic proteins, the feasibility crucially depends on the percentage of nuclei affected by paramagnetism.The solution structures of two 4Fe-4S high-potential ironsulfur proteins (Hipips), in both oxidation states, have been reCorrespondence...

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H‐NMR spectroscopy

“…The latter resonance is characterized by its NOE to a resonance at 4.24 ppm, presumably Asp 23 Ha. This assignment is supported by the fact that the structurally characterized cubane ferredoxins share a turn involving the fourth cysteine of the cluster binding sequence (C(IV)PXX) as a highly conserved structural element [16][17][18][19][20][21][22][23]. Owing to this turn, a strong NOE connectivity is expected between CysIV H(3 2 and the Ha of amino acid i+3 with respect to CysIV (Asp 23 in the present case).…”

“…The overall tertiary structure of B. schlegeliis ferredoxin maintains the pseudo 2-fold symmetry of all dicluster ferredoxins [19,21,22,24,25]. This is evident from the similarity of the ID spectra of the two proteins and the observation of the expected NOE between Hf$i of CysIII of cluster I and H(3i of CysIII of cluster II in both cases.…”

The Solution Structure Refinement of the Paramagnetic Reduced High‐Potential Iron‐Sulfur Protein I from Ectothiorhodospira Halophila by Using Stable Isotope Labeling and Nuclear Relaxation

“…This correction was made by recalculating the crosspeak intensities (ZJ through the relationship [ where pt" = pIJ is given by (4) where r,J is the internuclear I-J distance; y, is the nuclear magnetogyric ratio; o, the nuclear Larmor frequency (in rad SKI) and z, is the reorientational time constant for the two spin vectors. p r = pIM is given by the Solomon equation, (6) where y, is the nuclear magnetogyric ratio; r,, is the nucleusmetal distance; S is the electron spin angular momentum; and z, is the reorientational time constant for the nucleus-electron interaction. The latter is determined by the electron relaxation times.…”

The solution structure of paramagnetic metalloproteins

“…Many Fe-S proteins have then been studied with similar procedures, including the HiPIP from C. vinosum [34,35], the Fe 8 S 8 ferredoxin from C. pasteurianum [30,36] ferredoxins, the rubredoxin from C. pasteurianum [44], and some mutants of the above proteins [45,46]. Many paramagnetic cytochromes have also been studied (recently reviewed in [47]).…”