On the Production of Singlet Oxygen by the Isoalloxazine Ring in Free and Protein-bound Flavin Cofactors

Flavin cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), as a part of flavoenzymes play a critical role in the catalysis of multiple reactions predominantly of a redox nature. Question arises why nature developed two very similar cofactors with an identical functional part - isoalloxazine ring. We believe that an answer is related to the fact that the isoalloxazine ring belongs to endogenous photosensitizers able to produce reactive and potentially harmful singlet oxygen, O, with high efficiency, Φ ∼ 0.6. In fact, in contrast with one main conformation of FMN in water, the presence of the adenosine mononucleotide in FAD induces a dynamic equilibrium of two main conformations - closed (∼80 %) and open (∼20 %). The presence of predominant closed conformation of FAD in water has a significant impact on the Φ value, which is nearly 10-fold lower, Φ ∼ 0.07, than that of FMN. On the other hand, based on our analysis of a non-homologous dataset of FAD containing 105 proteins, ∼75 % enzyme-bound FAD exists predominantly in open conformations but the Φ values are significantly decreased, Φ < 0.03. We addressed these contradictory observations by analysis of: (i) dependence of Φ value on opening the FAD conformation by urea and (ii) amino acid propensities for isoalloxazine binding site. We demonstrated that urea-induced destabilization, in 7 M vs 0 M urea, of the closed FAD conformation leads to a ∼ 3-fold increase of Φ, proving the causative relation between Φ value and the flavin cofactor conformation. Detailed examination of the flavoproteins dataset clearly indicated positive propensities of three amino acids: glycine, cysteine, and tryptophan for isoalloxazine ring binding site. We hypothesize that both the closed conformation of free FAD and the arrangement of the isoalloxazine binding site is important for prevention of potentially harmful O production in cells.