Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe–S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity

Abstract

RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C methylthiolation of the D89 residue in the ribosomal S protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.

On the Role of Additional [4Fe-4S] Clusters with a Free Coordination Site in Radical-SAM Enzymes

“…Coordination of a [4Fe-4S] cluster by three cysteines and a HS − group has precedents in ( R )-2-Hydroxyisocaproyl-CoA dehydratase (Knauer et al, 2011) and HydG involved in maturation of hydrogenases (Dinis et al, 2015). CW electrochemistry suggested that the clusters have very close redox potentials (−420 mV vs. NHE) in the range typical of other RS enzymes (Pierrel et al, 2003; Molle et al, 2016). Moreover, a detailed EPR and Mössbauer study of RimO conducted in the absence of substrate indicated that addition of SAM to the reduced protein triggers a very fast re-oxidation of the RS cluster and none detectable products resulting from the reductolysis of SAM (Molle et al, 2016).…”

“…CW electrochemistry suggested that the clusters have very close redox potentials (−420 mV vs. NHE) in the range typical of other RS enzymes (Pierrel et al, 2003; Molle et al, 2016). Moreover, a detailed EPR and Mössbauer study of RimO conducted in the absence of substrate indicated that addition of SAM to the reduced protein triggers a very fast re-oxidation of the RS cluster and none detectable products resulting from the reductolysis of SAM (Molle et al, 2016). This event was accompanied on a longer time scale by the methylation of the hydrosulfide ligand bound to the UPF cluster (state C).…”

“…An alternative mechanism has proposed that the pentasulfide is methylated on its ω position (Landgraf et al, 2013) but this seems unlikely considering the constrained active site revealed by the X-ray structure (Forouhar et al, 2013). Thus, in the case of RimO, it appears that two molecules of SAM with different functions bind successively to the RS cluster and that this dual activity is under redox control (Molle et al, 2016). …”

“…Recently, DFT calculations performed on RimO led us to propose two additional steps completing the catalytic cycle (Molle et al, 2016). First, they showed that the reaction takes place only when the UPF cluster is in the oxidized state.…”

The Catalytic Mechanism of the Class C Radical S‐Adenosylmethionine Methyltransferase NosN

“…It has been shown that in the catalytic reactions of the methylthioltransferases MiaB and RimO,the methyl group is initially attached to the [4Fe-4S] cluster of the enzymes,a nd exogenous methanethiol can serve as the methyl source in the reaction. [14] However, in an assay with d 3 -SAM, NosN,and the other required components in the presence of methanethiol, unlabeled product was not produced in the reaction. This analysis excludes the possibility that NosN uses as trategy similar to MiaB and RimO in placing the methyl group on the [4Fe-4S] cluster before its transfer to the substrate.…”