The bacterial enzyme methylamine dehydrogenase (MADH) contains the protein-derived cofactor tryptophan tryptophylquinone (TTQ). TTQ is generated by the posttranslational modification of two endogenous MADH tryptophans, βW57 and βW108. Two oxygens are inserted sequentially into βW57 to generate the quinone moiety, after which it is cross-linked to βW108. We have previously shown that the second oxygenation and cross-link formation are catalyzed by the novel di-heme cytochrome MauG. Here we show, using isotopically labeled oxygen and water, that the first addition of oxygen occurs specifically at the C7 position of βW57 and that MauG then inserts the second oxygen at position C6.