Escherichia coli Biotin Synthase Produces Selenobiotin. Further Evidence of the Involvement of the [2Fe-2S]2+ Cluster in the Sulfur Insertion Step

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• 作者：Bernadette Tse Sum Bui ; Tony A. Mattioli ; Dominique Florentin ; Gé ; rard Bolbach ; and André ; e Marquet
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：March 21, 2006
• 年：2006
• 卷：45
• 期：11
• 页码：3824 - 3834
• 全文大小：151K
• 年卷期：v.45,no.11(March 21, 2006)
• ISSN：1520-4995

文摘

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotinbiosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin. The as-isolated enzymecontains a [2Fe-2S]²⁺ cluster, but the active enzyme requires an additional [4Fe-4S]²⁺ cluster, which isformed in the presence of Fe(NH₄)₂(SO₄)₂ and Na₂S in the in vitro assay. The role of the [4Fe-4S]²⁺cluster is to mediate the electron transfer to SAM, while the [2Fe-2S]²⁺ cluster is involved in the sulfurinsertion step. To investigate the selenium version of the reaction, we have depleted the enzyme of itsiron and sulfur and reconstituted the resulting apoprotein with FeCl₃ and Na₂Se to yield a [2Fe-2Se]²⁺cluster. This enzyme was assayed in vitro with Na₂Se in place of Na₂S to enable the formation of a[4Fe-4Se]²⁺ cluster. Selenobiotin was produced, but the activity was lower than that of the as-isolated[2Fe-2S]²⁺ enzyme in the presence of Na₂S. The [2Fe-2Se]²⁺ enzyme was additionally assayed withNa₂S, to reconstitute a [4Fe-4S]²⁺ cluster, in case the latter was more efficient than a [4Fe-4Se]²⁺ clusterfor the electron transfer. Indeed, the activity was improved, but in that case, a mixture of biotin andselenobiotin was produced. This was unexpected if one considers the [2Fe-2S]²⁺ center as the sulfursource (either as the ultimate donor or via another intermediate), unless some exchange of the chalcogenidehas taken place in the cluster. This latter point was seen in the resonance Raman spectrum of the reactedenzyme which clearly indicated the presence of both the [2Fe-2Se]²⁺ and [2Fe-2S]²⁺ clusters. No exchangewas observed in the absence of reaction. These observations bring supplementary proof that the [2Fe-2S]²⁺ cluster is implicated in the sulfur insertion step.