Inter- and intramolecular effects of tyrosyl residues on flavin triplets and radicals as investigated by flash photolysis.

Abstract— Addition of tyrosine or derivatives to aqueous solutions of flavins does not significantly impede either formation of the flavin triplet or the rate of O₂ oxidation of the flavin radical generated by reaction of triplet with the phenol. However, the rate of radical decay is decreased. There is only a modest effect that results from altering the nature of the group on alkyl side chains of the flavin when the substituent, e.g. phenylalanine, does not complex avidly with the isoalloxazine system. However, when a tyrosyl or O-methyltyrosyl residue is covalently attached to an alkyl side chain at the N¹⁰-position of the flavin, the considerable intramolecular complexing that results markedly decreases the formation of flavin triplet and, therefore, the radical yield. The rate of triplet decay is not much different than for noninternally complexed flavins, but extensive intramolecular radical decay occurs, and the rate of 0₂ oxidation of radical is decreased. A shorter alkyl chain is more effective than a longer one for decreasing triplet production, but the greater proximity of a photooxidiz-able tyrosyl residue to the flavin nucleus within the former allows a slightly higher intramolecular radical yield. Attachment of a tyrosyl residue by a short chain from the N³-position of the flavin has only a modest effect on the production of flavin triplet and its decay. There is less radical production from internal than from external tyrosyl residues, and the rate of O₂ oxidation of the flavin radical generated by such intermolecular photoreductants as N-acetyl tyrosine ethyl ester or EDTA is somewhat decreased. The tyrosyl residue within the active-site peptide of mitochondrial monoamine oxidase is not so susceptible to photooxidation by the 8α-(S-L-cysteinyl)flavin involved, since the thioether linkage at this position severely reduces triplet production. Upon oxidation of the thioether to sulfone, however, the triplet yield is partially restored. Some flavin radical can then be generated from either the intra- or an intermolecular tyrosyl residue. Taken together, these results demonstrate that tyrosyl residues near the flavin-binding sites of flavo-proteins can become oxidized by the flavin triplet that is light-generated unless the proximity and steric disposition of the interactants is such as to allow dissipation of much of the energy as radiationless decay within a tight complex or unless an 8α-thioether linkage to the flavin coenzyme is involved. Also, flavin radicals, whether generated photochemically or by biochemical oxidation of substrate, are readily oxidized by O₂ in the presence of tyrosyl functions unless tight complexing occurs. More remarkable, though, is the decreased rate of radical decay conferred by the association with a tyrosyl residue. This stabilization of reactive flavin radicals may have considerable consequence in the catalytic mechanism of such enzymes.