Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone( TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to beta Asp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-beta Asp-128 O-2 distance is similar to 3.7 angstrom, in contrast to the distance of similar to 2.7 angstrom predicted for the intermediate formed with the corresponding primary amine substrate. This difference of similar to 1.0 angstrom is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the similar to 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product.