Mechanism of Photoinduced Conformational Changes in the Photoenzyme Fatty Acid Photodecarboxylase Revealed by Light- Footprinting Ion Mobility Mass Spectrometry

Résumé fourni par la source

Fatty acid photodecarboxylase (FAP) converts fatty acids to hydrocarbons upon irradiation, rendering it one of the few known natural photoenzymes with potential for biofuel production. However, photoinactivation remains a major barrier for industrial applications. Here, we employ light footprinting ion mobility mass spectrometry (IMMS), together with complementary biophysical approaches, to investigate conformational changes and stability of FAP upon irradiation. Mass spectra of FAP reveal four major proteoforms: FAP is always bound to the FAD cofactor and up to two endogenous fatty acid substrates in the dark, whereas upon irradiation these non-covalent species are released to produce the apo form. Ion mobility data show an irradiation-induced conformational change in FAP concomitant with photoinactivation. Collision cross section distributions reveal the effect of fatty acid binding on the conformation and photoactivity of the protein: FAP adopts extended conformations under irradiation and a gradual decay in photoactivity. The unfolding of both apo-FAP and holo-FAP was examined using collisionally activating IMS (aIMS), which showed irradiation destabilizes the native conformations of holo-FAP. Multivariate analysis using IMMS data identified characteristic FAP analogues and FAD fragments distinguishing dark and irradiated states. The kinetics analysis of proteoform transitions were determined by light foot printing IMMS, revealing the events occurring during the productive and non-productive FAP photocycles at the proteoform and conformation levels. This study provides a new experimental approach for investigating photocatalytic and photoinactivation processes, and highlights the complex conformational dynamics required for efficient photocatalysis, which is crucial for understanding catalytic mechanism and guiding future protein engineering and design.

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