On the Origin of the Red-Shifted Flavin Absorption Spectra in Fatty Acid Photodecarboxylase

Fatty acid photodecarboxylase (FAP) is one of the few known natural photoenzymes and has attracted considerable interest due to its ability to convert fatty acids into hydrocarbons upon photoexcitation of its oxidized flavin adenine dinucleotide (FAD) cofactor. Notably, FAD in FAP exhibits an absorption spectrum red-shifted by approximately 10–15 nm compared to many other flavoproteins. This shift might arise from the specific electrostatics of the binding pocket and/or the slightly bent conformation of the FAD, as suggested by the crystallographic data. During the photocycle, an even more red-shifted intermediate (FADRS) has been observed, which ultimately reverts to the original state. In this work, we simulate the absorption spectrum of FAD inside FAP using a hybrid computational approach that combines quantum mechanics (QM) and molecular dynamics (MD) simulations in the Perturbed Matrix Method (PMM) framework. The computed absorption spectrum matches and explains the experimental one, not only validating the effectiveness of the MD-PMM approach but also revealing that the observed red shift primarily originates from the electrostatic environment provided by the protein matrix, whereas the effect of bending is comparatively minor. Additionally, we show that the formation of FADRS is unrelated to changes in active-site residue protonation or FAD conformation, but instead is likely to arise from a stable interaction between the flavin ring and bicarbonate, one of the proposed reaction products.