Solution Structure of the Oxidized Fe₇S₈ Ferredoxin from the Thermophilic Bacterium Bacillus schlegelii by ¹H NMR Spectroscopy^,

Abstract

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

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

“…Bs ferredoxin is known to be a thermostable protein. Temperature-dependent NMR studies on the WT showed that the protein can be heated up to 90°C for at least 15 min without appreciable denaturation or loss of the iron-sulfur clusters [11]. An analogous experiment was performed under anaerobic conditions on D13C, which was kept at 90°C for more than 25 min without noticeable denaturation (data not shown).…”

“…The structure calculations were carried out according to a recently developed torsion angle dynamics (TAD) approach using the program DYANA [20] as described earlier for the WT [11]. As in the latter case, they were supplemented by restrained energy minimization (REM) on the 20 structures of the DYANA family with the lowest target function values followed by restrained molecular dynamics (RMD) in vacuo.…”

“…The types of constraints employed for the present work have been described in detail earlier [11] : (i) dipolar connectivities from NOESY and 1D NOE difference spectra, (ii) distance constraints among the special Cys-S γ-Fe-S residues needed to assemble the two Fe 4 S 4 clusters as described for high-potential iron proteins [21] and C. pasteurianum 2[Fe 4 S 4 ] ferredoxin [22], (iii) constraints derived from hydrogen bonds, and (iv) dihedral angle constraints derived from the hyperfine shifts of cysteinyl βCH 2 protons [8,10].…”

“…The final DYANA family of 20 structures was refined with the SANDER [28] module of the AMBER 4.1 program package [29]. Details of the refinement procedure can be found elsewhere [11]. Additionally, the AMBER script makeCHIR RST was used to generate constraints that force the proper chirality on tetrahedral carbons and 'trans' constraints for all peptide bonds.…”

“…In the present work, we have solved the solution structure of the D13C mutant and compared it to that of the wild-type protein (WT) [11] in order to elucidate the structure of the mutant, to better assess the geometry of the newly formed cluster, to explore the possibility of long-range structural alterations that may originate from the mutation, and to shed more light on the folding problem of this class of proteins. It is worth recalling that ferredoxins do not fold in the absence of metals and therefore it is possible that even a difference in incorporation of one of the iron atoms may alter the folding pathway.…”