title: MiaB, a bifunctional radical-S-adenosylmethionine enzyme involved in the thiolation and methylation of tRNA, contains two essential [4Fe-4S] clusters.
abstract: 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 Mössbauer 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.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Hernández
authors.forename: Fabien
authors.initials: F
authors.lastname: Pierrel
authors.forename: Eric
authors.initials: E
authors.lastname: Elleingand
authors.forename: Ricardo
authors.initials: R
authors.lastname: García-Serres
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
authors.forename: Marc
authors.initials: M
authors.lastname: Fontecave
authors.forename: Mohamed
authors.initials: M
authors.lastname: Atta
mesh_terms: D002851:Chromatography, High Pressure Liquid; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008745:Methylation; D012343:RNA, Transfer / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D013438:Sulfhydryl Compounds; D012343:RNA, Transfer; C499584:MiaB protein, E coli; D013466:Sulfurtransferases