Enzymic synthesis of the 4Fe–4S clusters of Clostridium pasteurianum ferredoxin

Abstract

Ex novo enzymic synthesis of the two 4Fe-4S clusters of Clostridiumpasteuriunum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, DL-dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide.A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. (1) The nature of the cluster to be inserted into the reconstituted iron-sulfur protein is determined by the apoprotein itself. (2) The refolding of the structure of the iron-sulfur proteins around the newly inserted cluster is the rate-limiting step in both chemical and enzymic reconstitution. (3) Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron-sulfur protein(s).The extension to the 4Fe-4S centers of the rhodanese-based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron-sulfur structures.Clostridium pasteuriunum ferredoxin contains two ironsulfur clusters of the 4Fe-4S type and was the first iron-sulfur protein to be chemically reconstituted from its apoprotein, iron salts, thiols and sodium sulfide [I, 21. The same reconstitution procedure was applied to a number of iron-sulfur proteins with essentially good results, but the physiological meaning of these reactions remains doubtful.Enzymic synthesis of the 2Fe-2S cluster of spinach ferredoxin has been achieved by using the sulfurtransferase rhodanese and its substrates, thiosulfate and DL-dihydrolipoate, in place of the unphysiological sodium sulfide and thiols [3]. Rhodanese catalyzes the production of sulfide from thiosulfate with concomitant oxidation of one of the isomers of DL-dihydrolipoate to lipoate [4]. A productive interaction occurs between rhodanese and the apoprotein of spinach ferredoxin, favouring the process of cluster build-up and insertion [3]. In the reconstitution of spinach ferredoxin, the enzymic system appears to be regulated by the presence of the newly synthesized cluster on the protein to be reconstituted [5]. The same enzymic system was used recently to synthesize thiolate-substituted chemical analogues of the 2Fe-2S and of the 4Fe-4S centers [6].In order to verify whether the system based on the sulfurtransferase rhodanese can be thought of as a route for the in vivo synthesis of iron-sulfur structures, we have investigated the ex novo enzymic synthesis of the 4Fe-4S clusters of C. pasteurianum ferredoxin. The results are compared with those obtained in the chemical reconstitution of the same

Characterization of a 12-Kilodalton Rhodanese Encoded by glpE of Escherichia coli and Its Interaction with Thioredoxin

“…The iron-sulfur clusters of ferredoxins (10,51), succinate dehydrogenase (9), and mitochondrial NADH dehydrogenase (52) could be reconstituted by incubation with various combinations of bovine liver rhodanese, thiosulfate, dihydrolipoate, and iron ions. Interestingly, thioredoxin 1 has also been proposed to play a role in the formation or repair of iron-sulfur clusters (14).…”

“…Proposed roles include cyanide detoxification (61), sulfur metabolism (15,66), and mobilization of sulfur for iron-sulfur cluster biosynthesis or repair (9,10,51,52). At least two distinct rhodaneses and the related enzyme mercaptopyruvate sulfurtransferase have been described in E. coli, but the genes for these enzymes have not been mapped nor have the sequences for the proteins been determined (3,60,62).…”

Proteome-Wide Characterization of Seed Aging in Arabidopsis: A Comparison between Artificial and Natural Aging Protocols

“…However, the cyanide production from stored-seed glucosinolates has never been observed but should be investigated in the context of seed biology. Besides cyanide detoxification, proposed roles for sulfurtransferases are sulfur metabolism (Donadio et al, 1990) and the mobilization of sulfur for iron-sulfur cluster biosynthesis or repair (Pagani et al, 1984;Bonomi et al, 1985). Also, MST plays a physiological role in the protection against oxidative stress, and it particularly contributes to the maintenance of cellular redox homeostasis via the metabolic regulation of Cys degradation (Nagahara and Katayama, 2005).…”

Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

“…Subsequently, 100 mM LiS 2 was added dropwise to maintain the low overall sulfide concentrations and avoid precipitation of iron sulfides. [41] By this procedure iron ± sulfur clusters were formed and immediately incorporated into apo-Adx displayed at the bacterial surface. The successful folding of the Adx peptides around [2Fe ± 2S] clusters was indicated by a significant product formation in two cytochrome P450 enzyme tests (Figure 3).…”