How and why are some riboflavin coenzymes covalently attached to proteins?

Covalent flavinylation as elucidated by the formation of the histidyl(N3)-8 alpha-flavin bond in 6-hydroxy-D-nicotine oxidase of A. oxidans proceeds by an non-enzymatic mechanism. Incubation of the apoenzyme, FAD and a three-carbon phosphate ester at neutral pH leads to the formation of an enzymatically fully active holoenzyme. The role of His71 in this process was illustrated by site-directed mutagenesis. Nevertheless, the question whether covalent attachment of the cofactor in a holoenzyme has prevailed throughout the evolutionary screening process because of biological significance or whether it is a chance event of neutral selective value is still open.