Enzymic synthesis of the iron‐sulfur cluster of spinach ferredoxin

Abstract

A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80 7". Yields and kinetic parameters of the enzymic and chemical reconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build-up and insertion. The physiological relevance of this synthetic activity is discussed.Important progress has been made over the last few years in the field of iron-sulfur proteins. However, little still is known about the assembly of iron-sulfur clusters within the cell and their insertion into the various apoproteins.Attempts to reconstitute several iron-sulfur proteins by chemical methods have been more or less successful [1,2]. Because of the toxic nature of the reagents employed [3] and the non-specific chemical reactions [4], the physiological relevance of such models is doubtful.Evidence was also reported on the possible involvement of sulfurtransferascs, an ubiquitous class of enzymes [5,6], in the biosynthesis of iron-sulfur clusters [7,8]. 3-Mercaptopyruvate sulfurtransferase activity was found in both mitochondria and cytosol, but its involvement in the formation of the iron-sulfur cluster of adrenodoxin requires cysteine transaminase activity which is present almost only in the soluble fraction [7]. Rhodanese (thiosulfate-cyanide sulfurtransferase) activity was specifically found in mitochondrial fraction [9] and in chloroplasts where its activity appears to be related to active sulfur metabolism [lo].Rhodanese can restore chemical and functional properties, which have becn lost as a consequence of the alteration of the iron-sulfur cluster(s), in some iron-sulfur proteins such as mitochondrial succinate dehydrogenase [I 11 and NADH dehydrogenase [I21 as well as in the ferredoxins from either Clostridiumpasteurianum or spinach chloroplasts [I 31. A small amount of sulfur from radioactive thiosulfate was found inserted in the iron-sulfur protein as acid-labile [35S]sulfide [14,15]. The reducing equivalents which are necessary for the reduction of the sulfane sulfur of thiosulfate to sulfide were derived from the oxidation of sulfhydryl groups either on the iron-sulfur protein or on the sulfurtransferase itself [14 -161. In the latter case, a strong inactivation of rhodanese occurred. These results proved that rhodanese exerts a protective action on iron-sulfur proteins but they still do not provide direct evidence for an involvement of the sulfurtransferase in the exn o w synthesis of iron-sulfur clusters.Rhodanese is able to produce inorganic sulfide in the presence of its putative biological substrate, thiosulfatc, and of suitable dithiols, such as dihydrolipoate [I 71 o...

Pro108 is Important for Folding and Stabilization of Adrenal Ferredoxin, but does not Influence the Functional Properties of the Protein

“…This could indicate a possible scaffolding role for rhodanese in the reconstitution process starting from dilute protein solutions, as already suggested elsewhere [25]. As for other [FezS,] and [Fe,S,] ferredoxins [22,24], the rate of chemical reconstitution for either adrenodoxin mutant was independent of the sulfide concentration in the reconstitution medium (not shown). Thus, also for adrenodoxin the rate of the overall reconstitution process was determined by the acquisition of proper protein folding rather than by the velocity of cluster synthesis itself 122 -251.…”

Thiosulfate sulfurtransferase-like domain–containing 1 protein interacts with thioredoxin

“…Sulfurtransferases are ubiquitously distributed enzymes that function in a range of pathways including sulfur metabolism, iron-sulfur cluster formation, selenium metabolism, and cyanide detoxification (13,14,(25)(26)(27)(28). In humans, there are five sulfurtransferases including mitochondrial rhodanese and MST, which is found in the cytoplasmic and mitochondrial compartments (29,30).…”

“…Alternatively, the rhodanese domain can be present in a tandem repeat, as in rhodanese and MST, where the active sites are located in a cleft between the N-and C-terminal domains (14,15). Finally, rhodanese domains are fused to other protein domains as in Cdc25 phosphatase (20) or in the PRF and CstB proteins (23,24).Sulfurtransferases are ubiquitously distributed enzymes that function in a range of pathways including sulfur metabolism, iron-sulfur cluster formation, selenium metabolism, and cyanide detoxification (13,14,(25)(26)(27)(28). In humans, there are five sulfurtransferases including mitochondrial rhodanese and MST, which is found in the cytoplasmic and mitochondrial compartments (29,30).…”

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…Proposed roles include cyanide detoxification (Vennesland et al, 1982), sulfur metabolism (Donadio et al, 1990), and mobilization of sulfur for iron-sulfur cluster biosynthesis or repair (Bonomi et al, 1977;Pagani et al, 1984). In plants also the mobilization of sulfur for transport processes in older leaves was suggested (Papenbrock and Schmidt, 2000b).…”