Aromatic Residues Tune Excited-State Proton-Coupled Electron Transfer in BLUF Domains: Insights from CASPT2/MM Calculations

The blue light using flavin (BLUF) domain functions as a versatile photoreceptor switch mediated by proton-coupled electron transfer (PCET). Despite extensive ultrafast spectroscopic studies on BLUF mutants, the mechanistic role of key aromatic residues in regulating PCET pathways remains unclear. Here, we employ QM(MS-CASPT2)/MM calculations to elucidate the PCET mechanism of the FMN-Glu48-Trp6 (FMN-E-W) and FMN-Glu48-Tyr6 (FMN-E-Y) mutants. In the forward reaction, FMN-E-W follows a stepwise mechanism initiated by a PCET process from Glu48 to FMN, followed by another proton transfer from Trp6 to Glu48. In contrast, FMN-E-Y proceeds via a concerted PCET pathway without a stable intermediate. For the reverse process, FMN-E-W undergoes an electron transfer from FMN to Trp6 accompanied by a proton transfer to Trp6, followed by another proton transfer back to Glu48, whereas the reverse proton transfer in FMN-E-Y is both energetically and kinetically suppressed. These findings explain how aromatic residues tune PCET from stepwise to concerted, therefore supplying valuable insights for rationally designing photoresponsive proteins.