Rhodanese‐Mediated Sulfur Transfer to Succinate Dehydrogenase

Abstract

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].

Insertion of sulfide into ferredoxins catalyzed by rhodanese

“…The decay did not continue during storage at 2°C and in one case (table 2C) the ferredoxin-containing sample was inactivated less than the control. Ferredoxins differ in their effect on the catalytic efficiency of rhodanese; each reproduces some facet observed with succinate dehydrogenase [ 1]. The transferase was severely damaged in the interaction with spinach ferredoxin, as it is with the flavoprotein; with clostridial ferredoxin thiosulfate in the medium enhanced inactivation of rhodanese during reaction, and with both ferredoxins it made the reacted transferase less labile to chromatography.…”

“…It also competes with 3sS from thiosulfate in forming the sulfur-substituted rhodanese intermediate: indeed (unlabelled) rhodanese reacted with spinach ferredoxin appeared not to incorporate as much sulfur from thiosulfate as it could (up to 1 mol/mol) and as it does, for example, with succinate dehydrogenase [1 ]. It is interesting that this dilution did not occur with clostridial ferredoxin, where the impact of ferredoxinbound sulfane sulfur is most evident and where the (labelled) sulfur of rhodanese was diluted in sulfide formation.…”

“…After spectrophotometric assay the dye was extracted with butanol as described [ 1 ]. In samples containing thiosulfate or dithiothreitol the ZnS precipitate from inorganic and protein bound sulfide was separated by centrifugation and transformed into methylene blue according to [8].…”

“…We found that the sulfane sulfur of [3SS]rhodanese or that of thiosulfate was transferred to succinate dehydrogenase which bound it as sulfide and had its iron-sulfur structure modified. In a comparative assay with other proteins the presence of the iron-sulfur structure appeared important for sulfide binding [ 1 ].…”

“…In previous work [1] we studied the interaction of rhodanese (EC 2.8.1.1) an enzyme present in mitochondria, chloroplasts and bacteria, with the ironsulfur flavoprotein succinate dehydrogenase (EC 1.3.99.1). Rhodanese catalyzes the transfer of sulfane sulfur from a sulfane containing anion to a thiophilic anion.…”