Ultrafast photooxidation of protein-bound anionic flavin radicals

Abstract

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase

“…In the catalytic cycle of GOX, His516 is an important residue that has been described to participate in the concerted proton and hydride transfer in the reductive half‐reaction, [16,18] and has been demonstrated to be responsible for the oxygen activation in the oxidative half‐reaction [6,16] . The simulations of halide‐free GOX show that His516 is relatively flexible (Figure S12A and S11 A), consistent with previous computational studies [18,42] . During the F − ‐ and Cl − ‐containing simulations, both F − and Cl − interact with His516 when present in the GOX active site (Figure 6).…”

Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives

“…•− radical intermediates in the active site. Our recent study on a number of such oxidases shows that the Fl •− * excited state is extremely short-lived (<100 fs, Figure 4B) 4 to a point that time-resolved fluorescence could not be observed; previous reports on Fl •− * fluorescence 46,64 may have been complicated by admixture of minor quantities of other fluorescent forms of the flavin. Remarkably, in all flavin oxidases (including the GOX model system) transient absorption spectroscopy witnessed that Fl ox was formed quasi-instantaneously with high yield, demonstrating photooxidation of Fl •− (Figure 5).…”

“…Investigation of this hypothesis by timeresolved spectroscopic methods may shed light on such a mechanism, which should compete with cyclic photoinduced photooxidation of close-lying tyrosine and tryptophan residues occurring in ∼300 fs. 4 Interestingly, highly fluorescent flavin species cross-linked to the protein have also been reported in TrmFO, 55,56 and phototransformation of such an adduct has been observed. 56 Investigation of the photochemical processes occurring in complexes of flavins and protein residues is yet a virtually unexplored domain that may unearth new photocatalytic roles of flavins.…”

“…69,70 Intriguingly, the spectral properties of this band are very similar to the ones of the semireduced flavin anion Fl •− (Figure 1B), and the spectral properties of the initial photoproduct also show resemblance to those assigned to fully oxidized flavin in our recent photochemical study of flavoproteins containing semireduced flavin anion radicals. 4 Further theoretical and experimental investigations into such systems may help us to better characterize the electronic properties of the starting and intermediate states and ultimately devise strategies to further improve their photocatalytic properties.…”

“…The spectrum of FADH • is reproduced with permission from ref (copyright 1966 American Chemical Society) and normalized with respect to the spectrum of FAD ox . Adapted with permission from ref . Copyright 2022 the Authors.…”