Coenzyme F-420 is a redox cofactor found in methanogens and in various actinobacteria. Despite the major biological importance of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin, F-o) is still poorly understood. F-o synthase, the enzyme involved, is an unusual multidomain radical SAM enzyme that uses two separate 5'-deoxyadenosyl radicals to catalyze F-o formation. In this paper, we report a detailed mechanistic study on this complex enzyme that led us to identify (1) the hydrogen atoms abstracted from the substrate by the two radical SAM domains, (2) the second tyrosine-derived product, (3) the reaction product of the CofH-catalyzed reaction, (4) the demonstration that this product is a substrate for CofG, and (5) a stereochemical study that is consistent with the formation of a p-hydroxybenzyl radical at the CofH active site. These results enable us to propose a mechanism for F-o synthase and uncover a new catalytic motif in radical SAM enzymology involving the use of two 5'-deoxyadenosyl radicals to mediate the formation of a complex heterocycle.