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.