No 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...

The interaction of the sulfurtransferase rhodanese (EC 2.8.1 .l) with succinate dehydrogenase (EC 1.3.99.1), yeast alcohol dehydrogenase (EC 1.1.1.1) and bovine serum albumin was studied.Succinate Sulfur release from rhodanese appears to depend on the presence of -SH groups in the acceptor protein.Sulfur incorporated into succinate dehydrogenase was analytically determined as sulfide. A comparison of the optical spectra of succinate dehydrogenase preparations incubated with or without rhodanese indicates that there is an effect of the sulfurtransferase on the iron-sulfur absorption of the flavoprotein.The interaction of rhodanese with succinate dehydrogenase greatly decreases the catalytic activity of rhodanese with respect to thiocyanate formation. This is attributed to modifications in rhodanese associated with the reduction of sulfane sulfur to sulfide. Thiosulfate in part protects from this deactivation.The reconstitutive capacity of succinate dehydrogenase increased in parallel with sulfur incorporated in that enzyme following its interaction with rhodanese.Recent reports indicate that sulfurtransferases may be involved in sulfur transfer to iron-sulfur proteins. Taniguchi and Kimura [2] found that adrenodoxin could be reconstituted upon treatment of its apoprotein with 3-mercaptopyruvate and 3-mercaptopyruvate sulfurtransferase ; in another system, rhodanese (an enzyme that transfers sulfane sulfur) and thiosulfate (its substrate) substitute for inorganic sulfide in the reaction medium for restoring the iron-sulfur center of ferredoxin [3]. Details concerning the role of the sulfurtransferase enzyme in these reactions are not yet known.We have studied the interaction of rhodanese with the iron-sulfur flavoprotein succinate dehydrogenase. This paper is VII in a series on succinate dehydrogenase. The previous paper appeared elsewhere [I].

The affinity of aflatoxin M1 toward the main milk protein fractions in ewe and goat milk was investigated by using an ELISA. This study took into account the possible effects of common dairy processes such as ultrafiltration, acidic or rennet curding, and production of ricotta from acidic or rennet whey. Treatments that allowed the separation of casein from whey proteins under conditions that do not alter the physical or chemical status of the proteins (such as ultracentrifugation) were used as a reference. None of the treatments used in typical dairy processes caused significant release of the toxin, in spite of the relevant changes they induced in the interactions among proteins. Only the combined heat and acidic treatment used for production of ricotta cheese altered the structure of whey proteins to the point where they lost their ability to bind the toxin. This study also showed that, regardless of the physical state of the sample, a commercial electronic nose device, in combination with appropriate statistical tools, was able to discriminate among different levels of sample contamination.

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