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Received October 6, 2011; Revision received October 28, 2011
Iron–sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron–sulfur cluster biosynthesis. In this study, we cloned and purified iron–sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron–sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1 : 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron–sulfur cluster assembly; the cluster was well assembled at pH 7.6-8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron–sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe–2S] cluster-bound IscU under the optimal laboratory conditions.
KEY WORDS: iron–sulfur cluster, IscS, IscU, IscA-Fe, iron, cysteine