Incorporation of [35S]sulfide into the Fe-S cluster of aconitase.
Exchange of sulfide into the iron-sulfur cluster of beef heart aconitase was investigated using Na235S. After anaerobic incubation for minutes and up to 24 h, samples were freed of substances of low Mr by Sephadex G-50 and analyzed for protein, protein-bound Fe, S2-, total Fe-S cluster by EPR, and for radioactivity. The activated and the inactive enzyme exchange three S2- ions within 1-2 h at essentially equal rates. No further exchange is observed with the activated enzyme within 24 h, whereas with the inactive enzyme, (partial) exchange of one more S2- occurs slowly within 5-10 h. Exchange is facilitated at elevated pH, but then destruction of clusters also increases. During incubation of inactive enzyme with S2-, partial activation may occur because S2- can act as reductant. Thus, depending on conditions, we observed very low to substantial activities. There is a linear, positive correlation between activity developed and ratio of cluster bound Fe to S2-. When Fe and dithiothreitol are present together with S2- during incubation, the extent of S2-exchange generally is between 10 and 25%. Fe incorporation exceeds S2- exchange, with the difference between Fe and S2- incorporation consistently amounting to one Fe/cluster. It is suggested that this excess Fe represents the Fe ion taken up on completion of the [4Fe-4S] from the [3Fe-4S] structure. The ease of S2-exchange suggests that the Fe-S cluster of aconitase is readily accessible to solvent.
D000154:Aconitate Hydratase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis*; D007700:Kinetics; D008667:Metalloproteins / Q000096:biosynthesis*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D013440:Sulfides / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D013440:Sulfides; D013462:Sulfur Radioisotopes; D000154:Aconitate Hydratase