Ferredoxin II

The Mossbauer spectrum of the [4Fe-4S]- form of Dg Fd II consists of doublets 1 and 2 in a 1 3 intensity ratio (Fig. 9). When cluster conversion is carried out with 95% enriched Fe, the spectrum of the product consists of the more intense doublet 2 (132). Thus, in this case the externally supplied Fe occupies one or more of the three subsites b, which are equivalent by Mossbauer spectroscopy. Analysis of the Mossbauer spectra of the [4Fe-4S] cluster produced by dithionite reduction yielded results consistent with this picture. It was also established that the different subsites do not equilibrate at 25 °C over a time sufficient for protein reconstitution and cluster conversion. Oxidation of the [4Fe-4S] cluster with ferri-cyanide followed by examination of the product with EPR and Mossbauer spectroscopies proved transformation to the [3Fe-4S] form, thereby completing one cycle of cluster interconversion. 

The [3Fe-4S]/[4Fe-4S] cluster interconversion of aconitase is clearly subsite-specific because one and the same site is involved in the two reactions. The similar interconversion of Dg Fd II may be equally subsite-specific, but this cannot be proven. In this case, the three subsites b of the [4Fe-4S] cluster cannot actually be equivalent but are indistinguishable by the technique of resolution at hand, Mossbauer spectroscopy. What is 

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The ferredoxins isolated from D. gigas have been quite extensively studied by different experimental approaches and spectroscopic techniques and will be used here as a reference system. Ferredoxin I D. gigas Fdl) and ferredoxin II (D. gigas Fdll) (60-62) are composed of the same polypeptide chain (58 amino acids, 6 cysteines) (63). D. gigas Fdl is a dimer and contains a single [4Fe-4S], whereas the same monomeric unit of the tetrameric D. gigas Fdll contains a single [3Fe-4S] ° cluster. 

Fig. 7. EPR spectra of the [3Fe-4S] + center of D. gigas ferredoxin II recorded at 5 K in nonsaturating conditions at (a) 9 GHz Euid (b) 285 GHz. The 285 GHz spectrum was recorded at the Laboratoire des Champs Magn6tiques Intenses, CNRS, Grenoble.

Papaefthymiou, V., Girered, J.-J., Moura, I., Moura, J.J.G., and Miinck, E. 1987. Mossbauer study of D. gigas ferredoxin II and spi-coupling model for the Fe3S4 cluster with valence delocalization. Journal of the American Chemical Society 109 4703 1710. 

Ferredoxin II (Fd II) isolated from Desu ovibrio gigas is a small tetramer consisting of four identical subunits, each of molecular mass 6,000 daltons. Each subunit contains one FC3S4 cluster. A variety of studies have shown that the four clusters are identical and that they do not interaa with each other. Thus, for our purpose, we can consider Fd II as a monomer with one Fe3S4 cluster. 

We wish to acknowledge the contributions of Drs. Isabel Moura, Josd J. G. Moura and Jean LeGall who have worked with us for the past eight years on all projects involving ferredoxin II. This work was supported by a grant from the National Science Foundation and by a NATO Collaboration Research grant. 

Figure 16-18 Mossbauer X-ray absorption spectra of iron-sulfur clusters. (See Chapter 23 for a brief description of the method.) Quadrupole doublets are indicated by brackets and isomer shifts are marked by triangles. (A) [Fe2S2]1+ cluster of the Rieske protein from Pseudomonas mendocina, at temperature T = 200 K. (B) [Fe3S4]1+ state of D. gigas ferre-doxin II, T = 90 K. (C) [Fe3S4]° state of D. gigas ferredoxin II, T = 15 K. (D) [Fe4S4]2+ cluster of E. coli FNR protein, T = 4.2 K. (E) [Fe4S4]1+ cluster of E. coli sulfite reductase, T = 110 K. From Beinert et al.260...

Fig. 6.14. H NMR spectrum of oxidized D. gigas ferredoxin II [43], The two most downfield-shifted signals belong to a cysteine P-CH2 pair experiencing Curie-type temperature dependence.

Fig. 5. EPR spectrum of oxidized (native) D. gigas ferredoxin II, 0.180 mM in monomer. Spectrum recorded at the following instrument settings microwave power 30mW frequency 9.218 GHz modulation amplitude 4 G temperature 8 K and gain 1.250...

Fig. 6. Mossbauer spectrum of oxidized D. gigas ferredoxin II taken at 1.5 K in a field of 600 G applied parallel to the observed 7-radiation. The solid line plotted over the data is a superimposition of three simulated spectra describing components 1, 2 and 33s)...

Fig. 7a, b. Zero-field Mossbauer spectrum of D. gigas ferredoxin II... 

Fig. 8. EPR spectrum of reconstituted D. gigas ferredoxin II in the reduced form. The spectrum was recorded at the following instrumental settings microwave power 2 mW frequency 9.223 GHz modulation amplitude 10 G temperature 18 K and gain 1000...

The uper trace is the simulated Mossbauer spectra obtained using B. stearothermophilus [4 Fe-4 S] ferredoxin parameters for the reduced form46) and AEq and 6 values determined for reduced reconstituted D. gigas ferredoxin II. (Our unpublished results in collaboration with Drs. B.H. Huynh and E. Miinck)... 

C) oxidized and (D) reduced Scenedesmus Fe2S2 ferredoxin (2Fe + and [Fe, Fe ], respectively, data from Reference 348) (E) oxidized and (F) reduced Desulfovibrio gigas Fc3S4 ferredoxin II (3Fe + and [2Fe +, Fe +], respectively, data from Reference 158) (G) oxidized and (H) reduced Bacillus sterother-mophilus F64S4 ferredoxin (data from Reference 349). 

The resonance Raman spectrum of this complex bears a close resemblance to that of the D. gigas ferredoxin II. Since the vibrational bands responsible for the resonance Raman spectrum are not strongly dependent on the electronic properties, it is not surprising that an analogue with a different metal can be identified using this technique. 

The anion of [NEt4]3[Fe3S4(SPh)4] consists of two tetrahedral FeS4 units linked by a central Fe atom also in tetrahedral sites the two outer Fe atoms are each coordinated by two thiolate groups (structure 47). The Fe -Fe separation is 2.714 A, i.e. similar to that deduced from EXAFS experiments for D. gigas ferredoxin II. Despite this, the structures of the three-iron cores in the natural and synthetic systems are considered to be quite different as judged by their different ESR spectra (g 2.01 in the oxidized form of the protein, g x 4.2 in [Fe3S4(SEt)4l ") and other properties. 

Fig. 7b. Effect of oxidation and reduction on the epr spectra of M. flavum ferredoxins. (a) Ferredoxin II (0.59 mg/ml) as isolated in 25 mM Tris buffer pH 7.4 (b) after oxidation with 0.25 mM K3Fe(CN)e (c) after reduction with 0.2 mAf Na2S204 (d) ferredoxin I (0.66 mg/ml)>as isolated (e) after oxidation with 0.2 mM K FeCCNIe. Recording conditions were microwave frequency 9.200 GHz, temperature 12°K, field modulation 0.5 mT at 100 kHz and microwave power of 20 mW. The field bar corresponds to 10 mT. The arrow shows the magnetic field for g = 2.000 (from Yates e/o/., 1978).

Although the electron density for Cys-86 is much lower than those of other cysteinyl ligand residues, it should be noted that the side chain of Cys-86 is apparently rotated toward the solvent, away from the cluster, as observed for the nonligating cysteinyl residue (Cys ) of D. gigas ferredoxin II with a single cubane... 

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