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The radicalS-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

S/N crosstalk species derived from the interconnected reactivity of H₂S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe²⁺ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO⁻) and perthionitrite (SSNO⁻) to yield the dinitrosyl iron complex [Fe(NO)₂(S₅)]⁻. In the reaction of FeCl₂with SNO⁻we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl₂(NO)(SH)]⁻, which was characterized by X‐ray crystallography. We also show that [Fe(NO)₂(S₅)]⁻is a simple synthon for nitrosylated Fe−S clusters via its reduction with PPh₃to yield Roussin's Red Salt ([Fe₂S₂(NO)₄]²⁻), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe−S motifs.

S/N crosstalk species derived from the interconnected reactivity of H₂S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe²⁺ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO⁻) and perthionitrite (SSNO⁻) to yield the dinitrosyl iron complex [Fe(NO)₂(S₅)]⁻. In the reaction of FeCl₂with SNO⁻we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl₂(NO)(SH)]⁻, which was characterized by X‐ray crystallography. We also show that [Fe(NO)₂(S₅)]⁻is a simple synthon for nitrosylated Fe−S clusters via its reduction with PPh₃to yield Roussin's Red Salt ([Fe₂S₂(NO)₄]²⁻), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe−S motifs.