The iron-sulfur cluster (Fe4S4) centers in ferredoxins studied through proton and carbon hyperfine coupling. Sequence-specific assignments of cysteines in ferredoxins from Clostridium acidi urici and Clostridium pasteurianum

Abstract

Oxidized ferredoxin from Clostridium acidi urici, containing two [Fe4S4I2+ clusters, has been investigated through 1H NOESY and TOCSY spectroscopies. The protons of coordinated cysteines have been identified and assigned to each cluster with use of a procedure based on the assignment of two spatially close @CH2 pairs and on the shift ratios of each PCH2 proton in oxidized, half-reduced, and reduced forms; each cysteine proton has been then sequence-specifically and stereospecifically assigned by looking for dipolar connectivities with amino acid residues in the vicinity of the cluster. By comparing the present data with the available spectra of the analogous protein from Clostridium pasteurianum, the sequence-specific and stereospecific assignments of cysteine protons have been obtained also for the latter protein. The natural abundance 13C signals of the cysteine protons have been also sequencespecifically assigned. By taking advantage of the X-ray structure of a similar protein, the lH and I3C hyperfine shifts have been related to the dihedral angle between the iron-sulfur-0-carbon plane and the sulfur-fl-carbon-@-proton or sulfur-/3-carbon-a-carbon planes. A parametric equation is proposed. The spin delocalization mechanism has been found to be largely dependent on unpaired spin density on the pz orbital of the sulfur atom. Through EXSY spectroscopy, the proton signals of the [Fe&]+ clusters in the reduced protein have been assigned. Their temperature dependence is compared with that of the [Fe&I3+ clusters present in oxidized HiPIPs and discussed in terms of the Heisenberg model for the magnetic exchange coupling within the clusters.

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

“…As far as the hyperfine shifted resonances are concerned (Fig. 1), it is similar to the spectra of the 8Fe ferredoxins from Clostridium pasteurianum and C. acidi-urici [9], but different from the spectrum of Chromatium vinosum 8Fe ferredoxin [10]. The downfield region between 10 and 17 ppm contains numerous resonances (Fig.…”

“…a T, TOCSY cross-peak observed; N, NOESY cross-peak or ID NOE observed; aC, anti-Curie temperature dependence; ind, chemical shift independent of temperature; broad or sharp, qualitative line width in ID NOE difference spectra. Table 1 minus 2.8 ppm [9], Their relative relaxation rates R4/R2 were estimated from the linewidths in ID and ID NOE difference spectra. The figure depicts the relationship between Si and 82 for different dihedral angles, as represented by numbers in the ribbon [12].…”

“…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. On a more local structural level, we have applied to the D13C mutant a recently proposed approach to determine the Ca-CP-S-Fe dihedral angles of cysteines that coordinate [Fe4S 4 ] 2+ clusters from the chemical shifts and relaxation rates of their PCH 2 protons [9,12]. The dihedral angles of all cysteine residues have values that are compatible with the previously derived relationship between the hyperfine shifts (81, 8 2 ) of the PCH2 protons and their relative relaxation rates R1/R2 (Fig.…”

Sulfur K-Edge XAS and DFT Calculations on [Fe4S4]2+ Clusters: Effects of H-bonding and Structural Distortion on Covalency and Spin Topology

“…The magneto-structural correlation developed above suggests the same coordination mode in the resting [Fe 4 S 4 ] 2+ form (subclusters shaded red, coordinated by Cys14 and Cys8 and subcluster shaded blue, coordinated by Cys47 and Cys11, in Figure 3b). 41 The above correlations suggest that the orientation of the delocalized [Fe 2 S 2 ] + subclusters for both HiPIP and ferredoxins remains the same in all redox states, indicating that the oxidation or reduction process is primarily localized in one of the two [Fe 2 S 2 ] subclusters. In the case of ferredoxins, it is the solvent-exposed [Fe 2 S 2 ] + subcluster (Figure 3b, blue) that localizes the electron in the reduced state.…”

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

“…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.…”