Excited State Structure and Dynamics of the Neutral and Anionic Flavin Radical Revealed by Ultrafast Transient Mid-IR to Visible Spectroscopy

Abstract

Neutral and anionic flavin radicals are involved in numerous photochemical processes and play an essential part in forming the signaling state of various photoactive flavoproteins such as cryptochromes and BLUF domain proteins. A stable neutral radical flavin has been prepared for study in aqueous solution, and both neutral and anion radical states have been stabilized in the proteins flavodoxin and glucose oxidase. Ultrafast transient absorption measurements were performed in the visible and mid-infrared region in order to characterize the excited state dynamics and the excited and ground state vibrational spectra and to probe the effect of the protein matrix on them. These data are compared with the results of density functional theory calculations. Excited state decay dynamics were found to be a strong function of the protein matrix. The ultrafast electron transfer quenching mechanism of the excited flavin moiety in glucose oxidase is characterized by vibrational spectroscopy. Such data will be critical in the ongoing analysis of the photocycle of photoactive flavoproteins.

BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

“…We have previously reported vibrational mode assignments for FAD •– and FADH • ground states, and assigned transitions at 1528 cm –1 and 1626 cm –1 to the ground state of the radical (radical spectra are presented in SI Figure S4). 64 These radical marker modes complement those identified above for the neutral reactants. Although the 1626 cm –1 region in dAppA BLUF is crowded due to contributions from both protein and flavin, the 1528 cm –1 region is not and does not show the growth of an absorption which could be assigned to formation of FAD •– (Figure 2A); thus, there is no positive evidence for a radical state in the wild-type protein.…”

Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

“…34, 50 The improvement in signal quality of the current data enables us to clearly observe two features in this region at around 1515 and 1535 cm −1 (Figure 3A, B ), which have different kinetics (Figure 3D) and are assigned to FAD •− and FADH • , respectively, based on TRIR spectra of the anionic and neutral semiquinones formed by model flavins and glucose oxidase. 50 The 1515 cm −1 transient appears on a timescale of 2.5 ps and is assigned to formation of the FAD •− . As the transient at ~1515 cm −1 is decaying on the 20 ps timescale, a new transient is formed around 1535 cm −1 which we associate with the formation of FADH • by proton transfer (Figure 3A, B).…”

Short‐Lived Radical Intermediates in the Photochemistry of Glucose Oxidase

“…More recently, Lukacs et al. performed the first study on GOX using time‐resolved IR and visible absorption spectroscopy with full spectral resolution . Here, IR features associated with the rise in a few picoseconds and decay on the tens of picoseconds timescale were also tentatively ascribed to tryptophan radical intermediates.…”

“…At different wavelengths slower phases are clearly present, including those with opposing sign (490 nm), as also reported by others. [25,27] In the global analysis of the transient absorption data, a minimum of four phases were required, of which two had a time constant below 10 ps. Using 1-ps and 4-ps time constants retrieved from the fluorescence analysis, a satisfactory fit was obtained with an additional 37-ps phase and a long-lived phase; the corresponding species associated difference spectra (SADS) are shown in Figure 3 (right).…”