Journal
BIOCHEMISTRY
Volume 46, Issue 17, Pages 5140-5147Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi7000449
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- NIGMS NIH HHS [GM62542, GM47295] Funding Source: Medline
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The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79). The two [4Fe-4S](2+) clusters have similar UV-visible absorption, resonance Raman, and Mossbauer properties but differ in terms of redox properties and the EPR properties of the reduced [4Fe-4S](1+) clusters. Reconstituted forms of MiaB containing two [4Fe-4S] clusters are more active than previously reported. Comparison of MiaB with other radical-AdoMet enzymes involved in thiolation reactions, such as biotin synthase and lipoate synthase, is discussed as well as a possible role of the second cluster as a sacrificial S-donor in the MiaB-catalyzed reaction.
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