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Ultrafast Catalytic Mechanism and Molecular Dynamics of Fatty Acid Photodecarboxylase

Wu, Ruiqi
http://orcid.org/0000-0001-6848-0926
http://rave.ohiolink.edu/etdc/view?acc_num=osu1660904052656249

Abstract Details

2022, Doctor of Philosophy, Ohio State University, Biophysics.
Microalgal fatty acid photodecarboxylase (FAP) is a newly discovered flavin-bound photoenzyme that converts a carboxylic acid into a hydrocarbon and a carbon dioxide through decarboxylation. Unusually, the enzymatic reaction is driven by blue light. Since its discovery in 2017, FAP has aroused tremendous academic and industrial interests, as it has the potential to produce biofuel in a light-controlled manner. However, the catalytic mechanism of FAP is poorly understood. We carefully characterize its dynamic evolution with femtosecond spectroscopy and observed initial single electron transfer out of the substrate into the flavin cofactor in 347 ps with a stretched dynamic behavior, dominantly followed by a decarboxylation process in 5.8 ns to form an alkyl radical and a byproduct carbon dioxide. We also discovered a back electron transfer pathway that forms a futile cycle and compromises catalytic efficiency. We further identified the absorption bands of two radicals of carbonyloxy and alkyl intermediates at the active site. The overall enzymatic quantum efficiency is determined to be 0.81, consistently by either the obtained timescales or direct measurement, indicating a perfect hydrocarbon formation from the alkyl radical with the returned electron to close its photocycle. These results are essential to the elucidation of the enzyme mechanism and catalytic photocycle, providing a molecular basis for potential design of flavin-based photoenzymes. The catalytic mechanism involves an intermediate FAD•- state of the cofactor, but photochemical properties of this anionic radical are largely unknown. In this research, we have anaerobically produced and stabilized wild-type FAP in FAD•- state and conducted a series of steady-state and femtosecond-resolved spectroscopic investigations. Combining fluorescence and absorption measurements, we have identified conical intersections between potential energy surfaces of electronic states, which result in multiphase photoexcited FAD•-* decay in 1-2 ps, 50-95 ps and 1.2 ns. Interestingly, the nanosecond component can only form upon initial excitation to higher excited states. Our study demonstrates the deactivation of FAD•-* along complicated potential energy surfaces, and preliminarily rule out the occurrence of electron transfer or proton transfer.
Dongping Zhong (Advisor)
Jennifer Ottesen (Committee Member)
Adriana Dawes (Committee Member)
Kotaro Nakanishi (Committee Member)
Biophysics; Molecular Biology; Physical Chemistry
Fatty acid photodecarboxylase; photoenzyme; flavin; femtosecond dynamics; radical intermediates; photochemical mechanism

Recommended Citations

Citations

  • Wu, R. (2022). Ultrafast Catalytic Mechanism and Molecular Dynamics of Fatty Acid Photodecarboxylase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1660904052656249

    APA Style (7th edition)

  • Wu, Ruiqi. Ultrafast Catalytic Mechanism and Molecular Dynamics of Fatty Acid Photodecarboxylase. 2022. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1660904052656249.

    MLA Style (8th edition)

  • Wu, Ruiqi. "Ultrafast Catalytic Mechanism and Molecular Dynamics of Fatty Acid Photodecarboxylase." Doctoral dissertation, Ohio State University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=osu1660904052656249

    Chicago Manual of Style (17th edition)

osu1660904052656249
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This open access ETD is published by The Ohio State University and OhioLINK.