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Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

Significance The catalytic cycles of metalloenzymes comprise fleeting intermediates, which are challenging to trap and characterize. Their detection, however, can provide invaluable insights into reaction mechanisms. Herein, we focus on SuiB, a radical S-adenosylmethionine (rSAM) enzyme that introduces a carbon–carbon bond at unactivated positions onto its peptide substrate. We present evidence for an unprecedented intermediate, a transient lysine-cross-linked tryptophan radical and its oxidation by a SuiB-bound Fe-S cluster. Our studies provide evidence for a radical electrophilic aromatic substitution pathway, a mechanistic paradigm that is likely common in metalloenzyme-catalyzed carbon–carbon bond formations. They also carry broad implications for the 165,000 rSAM enzymes that contain auxiliary Fe-S clusters. The radical S-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.