Abstract
Upon blue-light absorption, LOV domains efficiently undergo intersystem crossing (ISC) to the triplet state. The efficiency of this ISC is said to arise from a heavy atom effect of the nearby (eventually adduct-forming) cysteine. However, LOV domain derivatives missing the cysteine residue still undergo ISC efficiently. Using hybrid multireference QM / MM models, we investigated the effect of the electrostatic environment in a prototypal LOV domain, AtLOV2, compared to the effect of the dielectric of an aqueous solution. We find that the electrostatic environment of AtLOV2 is especially well tuned to stabilize a triplet (nN,π*) state, which we posit is the state driving ISC. Other low-lying triplet states that have (π,π*) and (nO,π*) character are ruled out on the basis of energetics and / or orbital character. The mechanistic picture that emerges from the calculations is one that involves the ISC of photoexcited flavin to a (nN,π*) state followed by rapid internal conversion to a triplet (π,π*) state, which is the state detected spectroscopically. This insight into the ISC mechanism provides guidelines for tuning flavin's photophysics through mutations that alter the protein electrostatic environment and potentially helps explain why ISC does not occur readily in many classes of flavin-binding enzymes.