Ferredoxin isolation

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. 

Fe-4S] D. gigas Fdll structure is the only high-resolution crys-tallograph study performed in ferredoxins isolated from SRB (56)... 

The two ferredoxins isolated from Dsm. baculatum were investigated by EPR spectroscopy (67). The [4Fe-4S] center of Fdl shows a well-defined rhombic EPR signal in the reduced form at low temperature with g-values at 1.902, 1.937, and 2.068. The as-isolated Dsm. baculatum Fdll shows a weak contribution of a [3Fe-4S] + cluster (5%, Eo = 115 mV). Upon reduction a complex EPR signal appears,... 

Monomeric ferredoxins have been isolated and characterized from three methano-sarcinaceae [124-127] and one methanococcal species [128,129]. The methanosarcinal ferredoxins have primary sequences similar to clostridial 2x[4Fe-4S] ferredoxins but differ in the configuration of their [Fe-S] centers. Although the ferredoxins isolated from M. barkeri MS and M. barkeri Fusaro both have molecular masses of approximately 6 kDa they contain a [3Fe-3S] cluster and 2x[4Fe—4S] clusters, respectively. The ferredoxin from Methanosarcina thermophila has a molecular mass of only 4.9 kDa and contains either a [3Fe-3S] or a [4Fe-4S] center. These ferredoxins appear to be involved in electron transport from pyruvate dehydrogenase and from CODH [125,130]. A ferredoxin purified from Methanococcus thermolithotrophicus has 2x[4Fe—4S] centers, a molecular mass of 7.3 kDa and also appears to accept electrons from CODH [128,129]. 

The data analysis presented by Emptage et al.27) for the Av Fe-S III protein was complicated by the presence of two types of centers in the same molecule. However the novel center was also proven to be present in a ferredoxin isolated from D.gigas3S). This case represents a much simpler situation since the new type of center is the only core present. We will use this example as a prototype of proteins containing the three-iron center and its properties as representative of this new type of core. The center is referred as [3 Fe—xS] (see Table 1 and 3) due to the uncertainty on the number of labile sulfur present. We note that in aconitase (a protein which also contains this new center, as we will discuss later) equal amounts of iron and labile sulfur have been detected but the values obtained are quite low due to the presence of apoprotein68). 

Although we have focused on individual structural units in rubredoxins, ferredoxins and HIPIPs, we should note that some metalloproteins contain more than one Fe S unit. For example, the ferredoxin isolated from Azotobacter vinelandii contains both [4Fe S] and [3Fe-4S] units, with the closest Fe-"-Fe separation between units being i930 pm. 

Wada, K., Onda, M., and Matsubara, H. (1986). Ferredoxin isolated from plant non-photosyn-thetic tissues purification and characterization. Plant Cell Physiol. 27, 407-415. 

Four structural classes of Fe-S centre have been identified to date these are depicted in Fig. 13. All of them feature high-spin tetrahedral Fe(II) or Fe(III) coordinated typically by four sulfur donors. Apart from the monomeric centre found in proteins known as rubredoxins, they are all clusters that contain both protein donors and inorganic bridging (p) sulfido ligands. Most of our knowledge stems from studies made on the small electron-transport proteins known as ferredoxins (Fd s) and from work on model compounds. Figure 14 shows the structure of a ferredoxin isolated from the anaerobe Peptococcus aerogenes [165]. 

A number of proteins containing [3Fe-4S] centers bind certain metals in a reversible way to form cubane-iike clusters of the type [M,3Fe-4S]. The [3Fe-4S] core present in D.gigas Fdll was the first percursor used for the synthesis of hetero metal cores inside a protein matrix. Similar synthetic products have also been derived and extended with the ferredoxins isolated from Pyrococcus (P.) furiosus (39,47) and D.africanus (48). 

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