A Radical Intermediate in Bacillus subtilis QueE during Turnover with the Substrate Analogue 6-Carboxypterin

7-Carboxy-7-deazaguanine (CDG) synthase (QueE), a member of the radical S-deoxyadenosyl-l-methionine (SAM) superfamily of enzymes, catalyzes a radical-mediated ring rearrangement required to convert 6-carboxy-5,6,7,8-tetrahydropterin (CPH₄) into CDG, forming the 7-dezapurine precursor to all pyrrolopyrimidine metabolites. Members of the radical SAM superfamily bind SAM to a [4Fe-4S] cluster, leveraging the reductive cleavage of SAM by the cluster to produce a highly reactive 5'-deoxyadenosyl radical which initiates chemistry by H atom abstraction from the substrate. QueE has recently been shown to use 6-carboxypterin (6-CP) as an alternative substrate, forming 6-deoxyadenosylpterin as the product. This reaction has been proposed to occur by radical addition between 5'-dAdo· and 6-CP, which upon oxidative decarboxylation yields the modified pterin. Here, we present spectroscopic evidence for a 6-CP-dAdo radical. The structure of this intermediate is determined by characterizing its electronic structure by continuous wave and pulse electron paramagnetic resonance spectroscopy.