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

Aono, Shigetoshi; Bentrop, Detlef; Bertini, Ivano; Luchinat, Claudio; Macinai, Riccardo

doi:10.1016/s0014-5793(97)00834-x

“…The D13C variant of Bs Fe 7S8 protein shows hyperfineshifted signals for the βCH 2 protons of the coordinated cysteines that closely resemble those in native Fe 8 S 8 ferredoxins [8,10]. 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.…”

mentioning

confidence: 83%

“…The construction of the expression vector for the D13C mutant of Bs 7Fe ferredoxin was described earlier [8]. The mutant was expressed in E. coli and purified to homogeneity according to the procedure reported for WT [12].…”

Section: Methodsmentioning

confidence: 99%

“…They were processed with the standard Bruker software package and analyzed with the program XEASY [19]. The 1D NOE difference spectra obtained upon selective irradiation of the hyperfine-shifted resonances of the D13C variant of Bs ferredoxin were described earlier [8].…”

Section: Methodsmentioning

confidence: 99%

“…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].…”

Section: Methodsmentioning

confidence: 99%

“…An Asp13→Cys mutation in the Fe7S8 protein from Bacillus schlegelii (Bs hereafter) transformed this sequence motif into a Fe 4 S 4 -cluster-binding sequence [8]. However, an independent experiment to obtain a Fe 4S4 cluster with fourfold cysteine ligation by genetic engineering of a homologous Fe 3 S 4 cluster containing ferredoxin has been unsuccessful [9].…”

mentioning

confidence: 99%

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

Aono

¹

,

Bentrop

²

,

Bertini

³

et al. 1998

European Journal of Biochemistry

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The solution structure of the D13C variant of the thermostable Fe 7 S 8 ferredoxin from Bacillus schlegelii has been determined by 1 H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90°C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe 8 S 8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. 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 0.055 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 vicinity of the new cysteine, which becomes much more similar to native Fe 8 S 8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe 8 S 8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe 8 S 8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

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Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

Stout¹

2004

Handbook of Metalloproteins

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1

0

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This article reviews 7Fe ferredoxins (Fds), [FeS] proteins that contain [3Fe–4S] +/0 and [4Fe–4S] 2+/+ clusters. The 7Fe Fds from 19 prokaryotes are described in terms of biological function, and categorized with respect to cysteine ligand motifs, length of amino‐acid sequence, and metal ion content. Biochemical, mutagenesis, electrochemical, spectroscopic, and crystallographic data are summarized for well‐studied examples of distinct subclasses, including 7Fe Fds from Azotobacter vinelandii , Desulfovibrio africanus , Bacillus schlegelii , and Sulfolobus acidocaldarius . 7Fe Fds have been used to study cysteine ligand motifs, the interconversion of [3Fe–4S] and [4Fe–4S] clusters, proton transfer to [3Fe–4S] 0 clusters, and the protein control of [FeS] cluster reduction potential.

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Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

Stout

¹

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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This article reviews 7Fe ferredoxins (Fds), [FeS] proteins that contain [3Fe–4S] +/0 and [4Fe–4S] 2+/+ clusters. The 7Fe Fds from 19 prokaryotes are described in terms of biological function, and categorized with respect to cysteine ligand motifs, length of amino‐acid sequence, and metal ion content. Biochemical, mutagenesis, electrochemical, spectroscopic, and crystallographic data are summarized for well‐studied examples of distinct subclasses, including 7Fe Fds from Azotobacter vinelandii , Desulfovibrio africanus , Bacillus schlegelii , and Sulfolobus acidocaldarius . 7Fe Fds have been used to study cysteine ligand motifs, the interconversion of [3Fe–4S] and [4Fe–4S] clusters, proton transfer to [3Fe–4S] 0 clusters, and the protein control of [FeS] cluster reduction potential.

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

Cited by 11 publications

References 27 publications

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

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

Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

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

Cited by 11 publications

References 27 publications

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

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

Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

Ferredoxins Containing Two DifferentFe/SCenters of the Forms [4Fe–4S] and [3Fe–4S]

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Product

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

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

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