Effects of substrate length and active-site residue on catalytic function of fatty acid photodecarboxylase.

Fatty acid photodecarboxylases (FAPs) utilize blue light to convert fatty acids into Cn-1 hydrocarbons and CO2, providing a sustainable route to photobiocatalytic fuel production. Despite increasing interest, substrate-dependent phenomena-such as binding affinity, initial electron-transfer (ET) dynamics, and the influence of residues near the FAD cofactor-remain insufficiently characterized. Here, we systematically analyzed substrate binding, substrate-to-product conversion, and initial ET dynamics for three fatty acids of varying chain lengths using two cosolvent buffer systems. Ethanol improved substrate solubility but increased enzyme flexibility, leading to an extended FAD-substrate distance (DFAD-substrate) and diminished fluorescence quenching. Fluorescence titrations revealed chain-length-dependent binding, with palmitic acid (C16) and arachidic acid (C20) exhibiting stronger binding than lauric acid (C12). A fluorometric assay enabled quantification of product formation and catalytic half-lives, revealing that the fastest turnover arises from binding competition between substrate and product. Time-resolved fluorescence measurements further demonstrate that ET rates increase with substrate chain length. Longer fatty acids position their carboxylate groups closer to the FAD cofactor, reducing DFAD-substrate and accelerating the initial ET step. Finally, targeted mutagenesis at residue N170-located adjacent to the isoalloxazine ring-shows that local active-site interactions strongly modulate the partitioning of the excited 1FAD* pathways, thereby tuning photocatalytic efficiency and photostability.