Photophysics of Structurally Modified Flavin Derivatives in the Blue-Light Photoreceptor YtvA: A Combined Experimental and Theoretical Study

The light-induced processes of two flavin mononucleotide derivatives (1- and 5-deaza flavin mononucleotide, 1DFMN and 5DFMN), incorporated into the LOV domain of YtvA protein from Bacillus subtilis, were studied by a combination of experimental and computational methods. Quantum mechanics/molecular mechanics (QM/MM) calculations were carried out in which the QM part was treated by density functional theory (DFT) using the B3LYP functional for geometry optimizations and the DFT/MRCI method for spectroscopic properties, whereas the MM part was described by the CHARMM force field. 1DFMN is incorporated into the protein binding site, yielding a red-shifted absorption band (λ_max=530 nm compared to YtvA wild-type λ_max=445 nm), but does not undergo any LOV-typical photoreactions such as triplet and photoadduct formation. QM/MM computations confirmed the absence of a channel for triplet formation and located a radiation-free channel (through an S₁/S₀ conical intersection) along a hydrogen transfer path that might allow for fast deactivation. By contrast, 5DFMN-YtvA-LOV shows a blue-shifted absorption (λ_max=410 nm) and undergoes similar photochemical processes to FMN in the wild-type protein, both with regard to the photophysics and the formation of a photoadduct with a flavin-cysteinyl covalent bond. The QM/MM calculations predict a mechanism that involves hydrogen transfer in the T₁ state, followed by intersystem crossing and adduct formation in the S₀ state for the forward reaction. Experimentally, in contrast to wild-type YtvA, dark-state recovery in 5DFMN-YtvA-LOV is not thermally driven but can only be accomplished after absorption of a second photon by the photoadduct, again via the triplet state. The QM/MM calculations suggest a photochemical mechanism for dark-state recovery that is accessible only for the adduct with a C4a-S bond but not for alternative adducts with a C5-S bond.