Open Access
Arcinas, Arthur
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Doctor of Philosophy
Document Type:
Date of Defense:
December 05, 2016
Committee Members:
  • Squire J. Booker, Dissertation Advisor
  • Squire J. Booker, Committee Chair
  • Carsten D. Krebs, Committee Member
  • J. Martin Bollinger, Committee Member
  • Margherita T. Cantorna, Outside Member
  • J. Gregory Ferry, Committee Member
  • MiaB
  • methylthiolation
  • methylthiotransferase
  • Radical SAM
  • S-adenosylmethionine
  • iron-sulfur cluster
  • EPR spectroscopy
  • Mössbauer spectroscopy
  • ferrodoxin
  • electron film voltammetry
Methylthiolation (–SCH3) is a conserved tRNA modification found specifically in adenosines adjacent to the anticodon in tRNAs that decode a leading uridine. The resulting hypermodification confers improved anticodon-codon base pairing, resulting in greater accuracy and reading frame maintenance during translation The radical S-adenosyl-L-methionine (SAM) enzyme MiaB catalyzes the methylthiolation of the unactivated carbon 2 bearing an isopentenyl modification to their exocyclic nitrogen (i6A), to generate the 2-methylthio-N6- isopentenyladenosine group (ms2i6A). As a Radical SAM (RS) enzyme, MiaB harbors a canonical, redox active [4Fe–4S]2+/1+ RS cluster ligated at three Fe ions by cysteinyl residues within a typically conserved CX3CX2C motif, leaving an uncoordinated unique Fe site. In its reduced state ([4Fe–4S]1+), the RS cluster catalyzes the reductive cleavage of SAM to generate a 5’-deoxyadenosyl 5’-radical (5’–dA•) that abstracts a hydrogen radical (H•) from the C2 position of the substrate adenosine. A second, “auxiliary” [4Fe–4S] cluster (AC) in the UPF0004 domain of MiaB, shared among MTTases, also bears a unique Fe site similar to the RS cluster. As an MTTase, MiaB utilizes SAM as both a methyl donor, as well as a precursor to the 5’–dA• species. The methyl group in the methylthiol (–SCH3) modification is known to derive from SAM, a common methylating agent in the cell, and the sulfur is expected to originate from the AC in MiaB itself, and therefore be sacrificial in nature. The methylation of MiaB in the presence of SAM, a common methyl donor within the cell, was first detected by to the formation of S-adenosylhomocysteine (SAH), the product of methyl transfer. The formation of SAH, and methylation of MiaB by SAM, was determined to be independent of i6A tRNA substrate binding as well as reduction of the RS cluster to the [4Fe– 4S]1+ state by artificial chemical reductants such as sodium dithionite. MiaB methylation was observed using radioisotope labeling assays with [methyl-14C]SAM, which indicated the stoichiometric methylation of MiaB after gel filtration chromatography to separate protein from small molecules. Upon acid or base treatment of the methylated MiaB, the radioactivity was lost from the protein fraction and generated CH3SH gas. Time-dependent methylation of MiaB, acid quenched at specific times indicated the simultaneous formation of CH3SH and SAH, detected by GC-MS and LC/MS, respectively, at similar rates and final concentrations. The formation of CH3SH upon acid quenching MiaB incubated with SAM suggested that the methyl group acceptor from SAM is likely a [4Fe–4S] cluster of MiaB itself. Methanethiol was determined to be chemically competent as an intermediate species on the basis of its competitive incorporation into ms2i6A in MiaB assays incubated with CH3SH and reacted in the presence of [methyl- d3]SAM. Assays with MiaB incubated with SAM prior to catalyzing turnover in the presence of [methyl-d3]SAM generated ms2i6A during early time points and at faster rates than [methyl- d3]s2i6A, which contains a methyl group originating from the deuterated SAM. The concentration and rate of ms2i6A formation was coupled with that of 5’–dA-H, indicating the methylated MiaB intermediate was kinetically competent. The initial formation of ms2i6A prior to [methyl-d3]s2i6A suggested a single site for methyl transfer, and sequential methylation events. Based on the stoichiometry and final concentrations of natural abundance and deuterated products, a polysulfide species that is exogenously bound to the unique Fe site of the AC was proposed to be the methyl group acceptor from SAM. Methylthiolation by MiaB involves the cleavage of an unactivated C–H bond at the sp2- hybridized C2 center of the i6A substrate. The carbon site undergoing methylthiolation possesses a homolytic bond dissociation energy (HBDE) significantly higher than those of other bonds cleaved by the 5’–dA• radical. The investigation of whether the C2 site undergoes a hybridization change to sp3, or if a mechanism not involving direct abstraction of H• is operant, was pursued. MiaB assays conducted with [C2-d]i6A tRNA substrate demonstrated the stoichiometric formation of ms2i6A and deuterated 5’–dA (5’-dA-d) whereas assays using natural abundance i6A tRNA conducted in deuterium oxide (D2O) yielded ms2i6A coupled to 5’–dA formation. The formation of natural abundance 5’–dA and ms2i6A in experiments conducted in D2O indicates the H• abstracted by 5’–dA• originates from a non-solvent exchangeable position on the substrate, rather than a solvent-exchangeable heteroatom position. The C2 H• is therefore directly abstracted by 5’–dA• itself. A deuterium kinetic isotope effect of 1.84 was observed between 5’– dA-H and 5’-dA-d formation, indicating that H• abstraction in this position is at least partly rate limiting. Assays to trap substrate intermediates involved in nucleophilic addition to C2 utilized halogenated adenosine substrate analogs (2-Cl, 2-F)N6-isopentenyladenosine but failed to yield ms2i6A and 5’–dA. Additionally, attempts at observing radical substrate intermediate species by rapid freeze quench EPR also did not reveal any pertinent paramagnetic species. In addition to the origin of the methyl and sulfur moieties of methanethiol and the mechanistic details involved in its attachment to substrate, the in vivo reducing system that acts upon MiaB from the hyperthermophilic bacterium Thermotoga maritima (Tm) was investigated. Five ferredoxin genes (TM0927, TM1175, TM1289, TM1533 and TM1815) and the ferredoxin reductase (TM1639) from Tm were identified through sequence conservation and annotation, and were PCR amplified, ligated into an expression vector, overproduced and the resulting protein purified. The redox potential analysis of the ferredoxin Fe/S clusters, combined with sequence analysis, homology modeling and continuous wave electron paramagnetic resonance (CW-EPR) analysis were consistent with the presence of paramagnetic half-integer spin (S = 1⁄2) ground states of a [4Fe-4S]1+ cluster(s) upon dithionite reduction. Coupled activity assays with MiaB, TM1639 and the present ferredoxins support MiaB catalysis, and displayed decreased abortive cleavage of SAM to 5’–dA•, demonstrating improved coupling with the native reducing system. Protein Film Voltammetry (PFV), Electron Paramagnetic Resonance (EPR), Mössbauer spectroscopy and crystallography were utilized to characterize a [3Fe–4S] cluster species formed upon methylation of MiaB from the human gut bacterium Bacteroides thetaiotaomicron (Bt). PFV was utilized to determine that the redox potentials of the Bt MiaB clusters are centered at -425 mV with protein at rest, but one cluster undergoes a dramatic shift to -650 mV after incubation with SAM for 20 min. The formation of the low potential cluster could not be recapitulated with methanethiol, i6A tRNA substrate nor SAH. The assignment of the AC undergoing the negative potential shift conversion was consistent with EPR analysis indicating decreased signal amplitude for the paramagnetic S = 1⁄2 ground state signal of the cluster. The conversion of the AC from a cubane [4Fe–4S] species into a [3Fe–4S] form was identified in a preliminary crystal structure of Tm MiaB in complex with i6A tRNA and SAM. The ejected Fe ion was visible the structure and coordinated by conserved Asp and His residues among MTTases that catalyze methylthiolation of sp2 carbons (MiaB, MtaB). The [3Fe–4S] cluster species was confirmed by Mössbauer analysis indicating a field-strength dependent signal for a high–spin Fe3+ and a partially valence delocalized Fe2.5+ pair (37 % total Fe, ~1 cluster per MiaB) and the N/O coordinated high spin Fe2+ signal (12 % total Fe). Quantitation of the [3Fe–4S] cluster indicated a nearly complete conversion of the AC. The lack of significant paramagnetic S = 1⁄2 ground state [3Fe–4S]1+ signal assigns the cluster in the 0 oxidation state. Due to the similarities between the 3Fe4S cluster in MiaB with that observed in LipA, a reinvestigation of MiaB mechanism is necessary, in light of the potential mechanistic relevance of the cluster species.