Protein-Driven Electron-Transfer\nProcess in a Fatty\nAcid Photodecarboxylase

Naturally occurring photoenzymes are rare in nature,\nbut among\nthem, fatty acid photodecarboxylases derived from Chlorella\nvariabilis (<i>Cv</i>FAPs) have emerged\nas promising photobiocatalysts capable of performing the redox-neutral,\nlight-induced decarboxylation of free fatty acids (FAs) into C1-shortened <i>n-</i>alka(e)nes. Using a hybrid QM/MM approach combined with\na polarizable embedding scheme, we identify the structural changes\nof the active site and determine the energetic landscape of the forward\nelectron transfer (fET) from the FA substrate to the excited flavin\nadenine dinucleotide. We obtain a charge-transfer diradical structure\nwhere a water molecule rearranges spontaneously to form a H-bond interaction\nwith the excited flavin, while the FA’s carboxylate group twists\nand migrates away from it. Together, these structural modifications\nprovide the driving force necessary for the fET to proceed in a downhill\ndirection. Moreover, by examining the R451K mutant where the FA substrate\nis farther from the flavin core, we show that the marked reduction\nof the electronic coupling is counterbalanced by an increased driving\nforce, resulting in a fET lifetime similar to the WT, thereby suggesting\na resilience of the process to this mutation. Finally, through QM/MM\nmolecular dynamic simulations, we reveal that, following fET, the\ndecarboxylation of the FA radical occurs within tens of picoseconds,\novercoming an energy barrier of ∼0.1 eV. Overall, by providing\nan atomistic characterization of the photoactivation of <i>Cv</i>FAP, this work can be used for future protein engineering.