Protein-driven electron transfer process in a fatty acid photodecarboxylase

Naturally occurring photoenzymes are rare in nature, but among them, fatty acid photodecarboxylases derived from Chlorella variabilis (CvFAPs) have emerged as promising photobiocatalysts capable of performing redox-neutral, light-induced decarboxylation of free fatty acids (FAs) into C1-shortened n-alka(e)nes. This study focuses specifically on the protein-driven mechanism behind the initial forward electron transfer (fET) from the FA substrate to the excited flavin adenine dinucleotide (1FAD*), and the following decarboxylation in CvFAP. Using hybrid QM/MM approaches combined with a polarizable embedding scheme, we characterize the structural properties of the active site and explore the excited-state energy landscape of various scenarios involved in the fET process. We obtain an excited-state structure where a water molecule close to FAD rearranges spontaneously to form an H-bond interaction with FAD itself and, at the same time, the FA’s carboxylate group twists and migrates away from the excited flavin. Our findings indicate that it is the fET that naturally drives the cascade of structural rearrangements within the protein active site, which provide the necessary driving force for fET to proceed in a downhill manner and within a sub-ns timescale, given a significant interaction between the FA and the flavin. Additionally, QM/MM MD simulations reveal that following fET, the decarboxylation of the FA radical occurs within tens of ps, overcoming an energy barrier of ~0.1 eV. Overall, this work provides valuable insights into the fET and decarboxylation mechanisms, which are critical for determining the activity of FAPs and lay the groundwork for future protein engineering and design of novel flavin-based photoenzymes.