Fatty
acid photodecarboxylase (FAP) is a promising target for the
production of biofuels and fine chemicals. It contains a flavin adenine
dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation
of fatty acids to generate the corresponding alkane. However, little
is known about the catalytic mechanism of FAP, or how light is used
to drive enzymatic decarboxylation. Here, we have used a combination
of time-resolved and cryogenic trapping UV–visible absorption
spectroscopy to characterize a red-shifted flavin intermediate observed
in the catalytic cycle of FAP. We show that this intermediate can
form below the “glass transition” temperature of proteins,
whereas the subsequent decay of the species proceeds only at higher
temperatures, implying a role for protein motions in the decay of
the intermediate. Solvent isotope effect measurements, combined with
analyses of selected site-directed variants of FAP, suggest that the
formation of the red-shifted flavin species is directly coupled with
hydrogen atom transfer from a nearby active site cysteine residue,
yielding the final alkane product. Our study suggests that this cysteine
residue forms a thiolate-flavin charge-transfer species, which is
assigned as the red-shifted flavin intermediate. Taken together, our
data provide insights into light-dependent decarboxylase mechanisms
catalyzed by FAP and highlight important considerations in the (re)design
of flavin-based photoenzymes.
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