Protein Electrostatics Tune the Singlet–Triplet Energy Gap in Natural and Engineered Phototropin Light-Oxygen-Voltage (LOV) Domains

Photoactive flavoproteins such as Light-Oxygen-Voltage (LOV) domains serve as scaffolds for tuning the photophysics of their bound flavin cofactor through sequence mutations. This variable tuning effect has led to the development of a series of engineered LOV-based proteins that optimize fluorescence, intersystem crossing (ISC), photoreduction, and/or adduct formation over a range of time scales. To better guide future engineering efforts, we recently employed hybrid quantum mechanical/molecular mechanical (QM/MM) models of LOV domains to study how intradomain electrostatics exert control over flavin's photophysics. This work focuses on a series of three LOV1 and three LOV2 domains from <i>Arabidopsis thaliana</i> (AtLOV), <i>Avena sativa</i> (AsLOV), and <i>Chlamydomonas reinhardtii</i> (CrLOV), as well as two engineered protein variants used for singlet oxygen generation, miniSOG and SOPP3. The results, which include an analysis of conformational flexibility, energetics of low-lying singlet and triplet π,π* and <i>n</i>,π* states, and protein electrostatic projection maps, shed light on the variations in ISC efficiency in those systems. We found that LOV1 domains are more flexible than LOV2 domains and have consistently higher triplet <i>n</i>,π* <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>(</mml:mo><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:msub><mml:mo>)</mml:mo></mml:math> energies when compared to the first optically active singlet π,π* <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mo>(</mml:mo><mml:msub><mml:mrow><mml:mi>S</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mi>π</mml:mi><mml:mo>,</mml:mo><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:msub><mml:mo>)</mml:mo></mml:math> state. This finding corroborates reports in the literature that ISC is typically less efficient in LOV1 domains. We also find that unfavorable triplet state energetics may provide an alternative explanation for a competing adduct formation directly from the singlet <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mrow><mml:mi>S</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mi>π</mml:mi><mml:mo>,</mml:mo><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:msub></mml:math> state in CrLOV2. In contrast, SOPP3 was found to have a smaller energy gap between the <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mrow><mml:mi>S</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mi>π</mml:mi><mml:mo>,</mml:mo><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:msub></mml:math> and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msup><mml:mrow><mml:mi>π</mml:mi></mml:mrow><mml:mrow><mml:mo>*</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:msub></mml:math> compared to miniSOG and all other natural LOV domains, which explains its improved ability to sensitize triplet oxygen. Together, these results emphasize the importance of electrostatic tuning in controlling the efficiency of ISC in LOV domains. The results also indicate that a heavy-atom effect alone cannot explain efficient ISC, especially in Cys-devoid systems like SOPP3.