Metalloenzymes couple substrate binding and formation of oxidative intermediates to minimize unwanted side reactions. However, the molecular details of such coupling frequently remain ambiguous. Radical S-adenosylmethionine (SAM) enzymes constitute one of the largest groups of metalloenzymes and catalyze various radical-mediated reactions1. While radical SAM enzymes significantly accelerate the conserved radical initiation reaction, the reductive cleavage of SAM into 5′-deoxyadenosyl radical (5′-dA•)2, the molecular mechanism of the rate acceleration is significantly underexplored. Here, using the MoaA radical SAM enzyme in the molybdenum cofactor (Moco) biosynthesis3 as a model, we report the discovery of the mechanism of substrate-triggered radical initiation. We first solved the intact active site structure of MoaA (Figure 1) using solution NMR characterization of the C-terminal tail, which was disordered in the reported crystal structures, and its computational docking into the MoaA structure. We then investigated the structural elements critical for the radical initiation using functional characterization of mutant enzymes and peptides, HYSCORE, and DFT computation. The combined results suggest that MoaA uses the conformationally flexible C-terminal tail with two conserved Gly residues (GG motif) at the C-terminus as a sensor to detect substrate GTP binding and trigger reductive SAM cleavage. Importantly, mutations disrupting such a regulatory mechanism cause Moco deficiency disease in humans4. Comparison of these observations with other radical SAM enzymes provided insights into the general mechanism of substrate-triggered radical initiation in radical SAM enzymes.