8 alpha-(O-Tyrosyl)flavin adenine dinucleotide, the prosthetic group of bacterial p-cresol methylhydroxylase.

8 alpha-(O-Tyrosyl)riboflavin has been synthesized by condensation of the copper complex of L-tyrosine with 8 alpha-bromotetraacetylriboflavin. The structure of this synthetic product was proven by absorption and 1H NMR spectroscopy and by chemical degradation, which yielded 1 mol of tyrosine per mol of flavin. The synthetic compound comigrated wtih the (aminoacyl)riboflavin isolated from the p-cresol methylhydroxylase of Pseudomonas putida, and both showed identical absorption and fluorescence spectral properties. 8 alpha-(O-Tyrosyl)riboflavin as well as the flavin-containing decapeptide from p-cresol methylhydroxylase undergoes reductive cleavage to form riboflavin and FAD, respectively, on anaerobic treatment with dithionite. In contrast, the native enzyme, on reduction with dithionite, yields a reduced flavin via a red (anionic) flavosemiquinone intermediate, which remains covalently bound to the protein even under denaturing conditions. 8 alpha-(O-Tyrosyl)riboflavin bound to apoflavodoxin is also not cleaved on reduction with dithionite, but, instead, a blue (neutral) semiquinone of tyrosylriboflavin is generated, which is resistant to further reduction with dithionite. Three p-cresol methylhydroxylases, isolated from different strains of Pseudomonas putida, differing in molecular weight and Km values for substrates, contain the same peptide at the flavin site. These data provide definitive proof for the existence of 8 alpha-(O-tyrosyl)riboflavin in nature.