QM/MM Calculated Triplet–Singlet Infrared Difference Spectra of LOV2 Photoreceptor Domains and FMN in Solution

Abstract Light-oxygen-voltage (LOV) domains are photoreceptors that mediate critical cellular responses to blue light in plants, fungi, and bacteria. In the LOV2 domain of phototropin 1 from Avena sativa (AsLOV2), the primary photophysical event following photoexcitation of the flavin mononucleotide (FMN) chromophore is intersystem crossing to the triplet state. This is followed by formation of other transient intermediates leading to an adduct that triggers protein conformational changes. It is possible to follow the formation and decay of those intermediates and conformational states using methods like time-resolved (TR) Fourier-transform infrared (FTIR) difference spectroscopy (DS). The interpretation of TR-FTIR DS can be aided by computational models that connect experimental signals with molecular modes that describe the chemistry of the system. In this work, a hybrid quantum mechanics/molecular mechanics (QM/MM) protocol is employed to calculate the triplet–singlet (T–S) TR-FTIR DS of FMN within AsLOV2 and for free FMN in water. For AsLOV2, snapshots from molecular dynamics (MD) simulations are selected to carry out QM/MM vibrational frequency calculations. Only a few MD calculated structures need to be sampled for the protein to produce (T–S) FTIR DS that are in good agreement with experiment. In comparison, considerably more sampling is required for flavin–water geometries to simulate the (T–S) FTIR DS of free FMN in solution due to the increased randomness of solvent interactions. The calculated spectra correctly capture the frequency shifts associated with the change in flavin’s electronic structure in the triplet state for both the protein and for free FMN in solution relative to the singlet. Deviations from the experimental frequencies may be explained in part by mixing between the lowest (π,π*) triplet state of FMN and the second (n,π*) triplet state, in accordance with recent reports in the literature of such mixing in free and protein-bound FMN.