Light‐generated Paramagnetic Intermediates in BLUF Domains^†

Abstract

Blue-light sensitive photoreceptory BLUF domains are flavoproteins, which regulate various, mostly stress-related processes in bacteria and eukaryotes. The photoreactivity of the flavin adenine dinucleotide (FAD) cofactor in three BLUF domains from Rhodobacter sphaeroides, Synechocystis sp. PCC 6803 and Escherichia coli have been studied at low temperature using time-resolved electron paramagnetic resonance. Photoinduced flavin triplet states and radical-pair species have been detected on a microsecond time scale. Differences in the electronic structures of the FAD cofactors as reflected by altered zero-field splitting parameters of the triplet states could be correlated with changes in the amino-acid composition of the various BLUF domains' cofactor binding pockets. For the generation of the light-induced, spin-correlated radical-pair species in the BLUF domain from Synechocystis sp. PCC 6803, a tyrosine residue near the flavin's isoalloxazine moiety plays a critical role.

One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein

“…A comparable effect has been observed in the triplet state of a methionine mutant of the photoreceptor YcgF. 61 Continuous-Wave EPR Spectroscopy. Pulsed and continuous-wave paramagnetic electron resonance spectroscopy is a widely used sensitive method to detect radicals and other paramagnetic molecules.…”

“…59 Following the analysis outlined previously, 61 the spectra show a characteristic powder pattern centered around the g value of the flavin triplet state that is dependent on the two zero-field splitting parameters D and E and the populations p i with i = x, y, z of the three triplet sublevels. In general, all samples except Rf-LumP T50W show a trEPR signal that can be fully described by a flavin triplet state.…”

Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors

“…The light-induced reaction produced various spectrally distinct radical pair difference spectra on the ultrafast time scale, which were assigned to FAD/Trp-8 radical pairs but might also correspond to a radical pair consisting of FAD and the semiconserved Trp-91. Using transient EPR spectroscopy, a strongly coupled radical pair was detected in this mutant, which was significantly different from the WT radical pair and decayed slightly slower (74). Because the Y8W mutation, however, rendered the BLUF photoreceptor non-functional and the radical pairs probably originate from the FAD triplet state rather than the singlet excited state, these findings are difficult to relate to WT and functional BLUF mutants directly.…”

“…Previously, we modulated the tyrosine redox partner by exchanging the phenolic side chain by an indole moiety using site-directed mutagenesis (44,74). Although the Slr1694-Y8W mutant was able to photoreduce the flavin to a radical state with high efficiency, no signaling state was formed, probably due to alterations in the flavin-coordinating hydrogen bond network involving Gln-50.…”

Molecular eyes: proteins that transform light into biological information

“…Accordingly, the removal of the conserved glutamine prevents signalling state formation [25], although a neutral flavin semiquinone is formed in the Slr1694 Q50A mutant that lives for a few nanoseconds (T. Mathes & R. Fudim 2011, unpublished data). By replacement of the phenol side chain by an indole (Y8W), which, in principle, should provide a similar chemical basis to both donate electrons and protons to the flavin-like tyrosine, we also observed the formation of a mixture of several flavin radical pair species, which however also did not yield the red-shifted signalling state [70,71]. These studies showed that not only light-induced PCET is required for BLUF photoactivation but also defined interactions between protein and flavin are necessary for the hydrogen bond switch to be facilitated.…”