Copper amine oxidase contains an organic redox cofactor, 2,4, 5-trihydroxyphenylalaninequinone (topaquinone, TPQ), derived by the post-translational modification of a specific tyrosyl residue. To identify amino acid residues participating in the biogenesis of TPQ in the recombinant phenylethylamine oxidase from Arthrobacter globiformis, we have modified the copper/TPQ-less apoenzyme and the copper/TPQ-containing holoenzyme with 4-fluoro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-F). In the apoenzyme modification, the Cu2+-dependent, self-processing formation of the TPQ cofactor was retarded in accordance with the amount of NBD incorporated. The holoenzyme was also rapidly inactivated by incubation with NBD-F. The inactivation was prevented almost completely in the presence of an oxidation product from phenylethylamine, phenylacetaldehyde. Furthermore, the reaction of an inhibitor, phenylhydrazine, with TPQ was much slower in the NBD-labeled holoenzyme than in the native holoenzyme. Sequence analysis of the NBD-labeled holoenzyme has identified Lys184 and Lys354 as the labeled sites. The two Lys residues are located close to the entrance to a channel, which has been found by recent X-ray crystallographic studies to be suitable for the movement of substrates and products to and from the Cu2+/TPQ-active site buried in the protein interior (Wilce, M. C. J., et al. (1997) Biochemistry 36, 16116-16133). However, site-specific mutant enzymes for Lys184, Lys354, and the neighboring invariant His355 had normal capacities for the TPQ formation in apoenzyme. These residues were also found to be dispensable for catalytic activity of holoenzyme. Thus, modification of Lys184 and Lys354 with NBD-F presumably causes structural perturbations of the substrate channel or steric hindrance for the access of small molecules to the active site through the channel.