Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallest ␥-subunit of the ␣␤␥ heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the ␣-and ␥-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The ␣-and ␤-subunits were both detected in the periplasm of the mutant strain, whereas the ␥-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the ␥-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster-and SAM-binding ORF2 protein is necessary for the posttranslational processing of ␥-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links. Quinohemoprotein amine dehydrogenase (QHNDH)4 is produced in the periplasmic space of certain Gram-negative bacteria, such as Paracoccus denitrificans and Pseudomonas putida, in response to primary amines, including n-butylamine and benzylamine, added to the culture medium as a sole carbon and energy source and catalyzes their oxidative deamination (1-3). QHNDH contains a posttranslationally derived quinone cofactor, cysteine tryptophylquinone (CTQ) (Fig. 1A), within the smallest ␥-subunit of the ␣␤␥ heterotrimeric protein (4 -6). The largest ␣-subunit folds into four domains containing two c-type hemes in the first domain (5, 6). The ␤-subunit has a seven-bladed ␤-propeller structure often observed in quinoproteins. CTQ is the most recently identified, fourth quinone cofactor derived from an amino acid residue(s) constituting the polypeptide chain after the first identified topa quinone of copper amine oxidase (7), the second identified tryptophan tryptophylquinone (TTQ) (Fig. 1B) of methylamine dehydrogenase (MADH) (8), and the third identified lysine tyrosylquinone of lysyl oxidase (9). In addition to CTQ that is essential as a redox cofactor for the catalytic reaction (1...