Ferredoxins as interchangeable redox components in support of MiaB, a radical S-adenosylmethionine methylthiotransferase.

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.

Parsing redox potentials of five ferredoxins found within Thermotoga maritima.

Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).

Determining Redox Potentials of the Iron-Sulfur Clusters of the AdoMet Radical Enzyme Superfamily.

While protein film electrochemistry (PFE) has proven to be an effective tool in the interrogation of redox cofactors and assessing the electrocatalytic activity of many different enzymes, recently it has been proven to be useful for the study of the redox potentials of the cofactors of AdoMet radical enzymes (AREs). In this chapter, we review the challenges and opportunities of examining the redox cofactors of AREs in a high level of detail, particularly for the deconvolution of redox potentials of multiple cofactors. We comment on how to best assess the electroactive nature of any given ARE, and we see that when applied well, PFE allows for not only determining redox potentials, but also determining proton-coupling and ligand-binding phenomena in the ARE superfamily.

Transformations of the FeS Clusters of the Methylthiotransferases MiaB and RimO, Detected by Direct Electrochemistry.

The methylthiotransferases (MTTases) represent a subfamily of the S-adenosylmethionine (AdoMet) radical superfamily of enzymes that catalyze the attachment of a methylthioether (-SCH3) moiety on unactivated carbon centers. These enzymes contain two [4Fe-4S] clusters, one of which participates in the reductive fragmentation of AdoMet to generate a 5'-deoxyadenosyl 5'-radical and the other of which, termed the auxiliary cluster, is believed to play a central role in constructing the methylthio group and attaching it to the substrate. Because the redox properties of the bound cofactors within the AdoMet radical superfamily are so poorly understood, we have examined two MTTases in parallel, MiaB and RimO, using protein electrochemistry. We resolve the redox potentials of each [4Fe-4S] cluster, show that the auxiliary cluster has a potential higher than that of the AdoMet-binding cluster, and demonstrate that upon incubation of either enzyme with AdoMet, a unique low-potential state of the enzyme emerges. Our results are consistent with a mechanism whereby the auxiliary cluster is transiently methylated during substrate methylthiolation.

Molecular basis of cobalamin-dependent RNA modification.

Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis Our structure of QueG bound to a tRNATyr anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.

Spectroscopic and Electrochemical Characterization of the Iron-Sulfur and Cobalamin Cofactors of TsrM, an Unusual Radical S-Adenosylmethionine Methylase.

TsrM, an annotated radical S-adenosylmethionine (SAM) enzyme, catalyzes the methylation of carbon 2 of the indole ring of L-tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from SAM; however, the enzyme also requires cobalamin and iron-sulfur cluster cofactors for turnover. In this work we report the overproduction and purification of TsrM and the characterization of its metallocofactors by UV-visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies as well as protein-film electrochemistry (PFE). The enzyme contains 1 equiv of its cobalamin cofactor in its as-isolated state and can be reconstituted with iron and sulfide to contain one [4Fe-4S] cluster with a site-differentiated Fe(2+)/Fe(3+) pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five-coordinate base-off/His-off conformation, whereby the dimethylbenzimidazole group is replaced by a non-nitrogenous ligand, which is likely a water molecule. Electrochemical analysis of the protein by PFE indicates a one-electron redox feature with a midpoint potential of -550 mV, which is assigned to a [4Fe-4S](2+)/[4Fe-4S](+) redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron site of the cluster in the same manner as in other radical SAM (RS) enzymes, yet its binding still perturbs the electronic configuration of both the Fe/S cluster and the cob(II)alamin cofactors. These biophysical studies suggest that TsrM is an atypical RS enzyme, consistent with its reported inability to catalyze formation of a 5'-deoxyadenosyl 5'-radical.

Electrochemical Resolution of the [4Fe-4S] Centers of the AdoMet Radical Enzyme BtrN: Evidence of Proton Coupling and an Unusual, Low-Potential Auxiliary Cluster.

The S-adenosylmethionine (AdoMet) radical superfamily of enzymes includes over 113,500 unique members, each of which contains one indispensable iron-sulfur (FeS) cluster that is required to generate a 5'-deoxyadenosyl 5'-radical intermediate during catalysis. Enzymes within several subgroups of the superfamily, however, have been found to contain one or more additional FeS clusters. While these additional clusters are absolutely essential for enzyme activity, their exact roles in the function and/or mechanism of action of many of the enzymes are at best speculative, indicating a need to develop methods to characterize and study these clusters in more detail. Here, BtrN, an AdoMet radical dehydrogenase that catalyzes the two-electron oxidation of 2-deoxy-scyllo-inosamine to amino-dideoxy-scyllo-inosose, an intermediate in the biosynthesis of 2-deoxystreptamine antibiotics, is examined through direct electrochemistry, where the potential of both its AdoMet radical and auxiliary [4Fe-4S] clusters can be measured simultaneously. We find that the AdoMet radical cluster exhibits a midpoint potential of -510 mV, while the auxiliary cluster exhibits a midpoint potential of -765 mV, to our knowledge the lowest [4Fe-4S](2+/+) potential to be determined to date. The impact of AdoMet binding and the pH dependence of catalysis are also quantitatively observed. These data show that direct electrochemical methods can be used to further elucidate the chemistry of the burgeoning AdoMet radical superfamily in the future.