The contribution of (8α)-(N₃) histidyl bond formation to the conformation of covalently flavinylated proteins was investigated by trypsin treatment of wild type and mutant versions of a model enzyme, 6-hydroxy-D-nicotine oxidase (6-HDNO) of Arthrobacter nicotinovorans. In the absence of FAD, apo-6-HDNO exhibited a conformation exposing a protease accessible site. Holoenzyme formation through FAD-attachment to His₇₁ induced a conformational change in the protein that shielded the trypsin recognition site. This conformational change, however, did not require FAD-histidyl bond formation since trypsin resistance was also exhibited by a 6-HDNO•Cys₇₁ mutant protein which was unable to bind FAD covalently. Replacement of Arg₆₇, an amino acid residue supposed to be essential in flavinylation, by Ala rendered the protein protease sensitive as did replacement of Pro₇₃ by Ala. These amino acids apparently play an essential role in stabilizing the native protein conformation. The inability to reach the native conformation also prevented FAD attachment, indicating that a specific conformation of the protein is a prerequisite for FAD-histidyl bond formation. Deletion of Phe₄₄₈ and Arg₄₄₉ from the 458 amino acid residuescontaining enzyme resulted in complete protease sensitivity, demonstrating that flavinylation takes place posttranslationally.