The path to the triplet state in LOV domains

Upon blue light absorption, LOV domains efficiently undergo intersystem crossing (ISC) to the triplet state. Several factors potentially contribute to this efficiency. One often proposed in the literature is the heavy atom effect from the nearby (and eventually adduct-forming) cysteine. However, some LOV domain derivatives missing the cysteine residue also undergo ISC efficiently. Using hybrid multi-reference quantum mechanical / molecular mechanical (QM/MM) models, we investigate the effect of the protein electrostatic environment in a prototypal LOV domain, AtLOV2, compared to the effect of the dielectric of a solvent. We find that the AtLOV2's electrostatic environment is especially well tuned to stabilize a triplet (nN,π*) state, which we posit is the state involved in the ISC step. Other low-lying triplet states having (π,π*) and (nO,π*) character are ruled out based on energetics and/or their orbital shape. The mechanistic picture that emerges from the calculations is one that involves ISC of photoexcited flavin to a triplet (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 can provide guidelines for tuning flavin's photophysics through mutations that alter the protein electrostatic environment and potentially helps explain why ISC (and subsequent flavin photochemistry) doesn't occur more frequently in many classes of flavin-binding enzymes