Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines. The enzyme from Pseudomonas putida has an αβγ heterotrimeric structure with two heme c groups in the largest α subunit, and a novel quinone cofactor [cysteine tryptophylquinone (CTQ)] and hitherto unknown internal cross-bridges in the smallest γ subunit. The crystal structure of the enzyme in the complex with the inhibitor [p-nitrophenylhydrazine (pNPH)] has been determined at a 2.0 Å resolution. 1 1Coordinates have been deposited in the Protein Data Bank (ID code 1JMZ). The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor. The Asp33γ is unambiguously determined as the catalytic base to abstract the α-proton from a substrate, because Nβ atom of the inhibitor corresponding to the Cα atom of the substrate amine is neighbored to Asp33γ. The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the β- and γ-subunits. The cofactor-inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component. The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site. The inhibitor may have access to the active site pocket through the water-filled crevice between the β- and γ-subunits.