On the Role of Aromatic Side Chains in the Photoactivation of BLUF Domains

Gauden, Magdalena; Grinstead, Jeffrey S.; Laan, Wouter; Stokkum, Ivo H. M. van; Ávila-Pérez, Marcela; Toh, K.C.; Boelens, Rolf; Kaptein, Robert; Grondelle, Rienk van; Hellingwerf, Klaas J.; Kennis, John T. M.

doi:10.1021/bi7006433

Cited by 110 publications

(168 citation statements)

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“…In the last, nondecaying EADS (magenta line), all signs of FAD* or FAD radical species have disappeared, and the difference spectrum corresponds to the red-shifted photoactivated state of BLUF domains, BLUF RED , apart from a low-amplitude, broad absorption between 550 and 680 nm. The latter may correspond to a small fraction of FAD triplet states, as observed previously in the AppA BLUF domain (20,35). The overall spectral evolution of the W91F mutant in H 2 O and D 2 O is quite similar to that of wild-type Slr1694 (19,21).…”

Section: Resultssupporting

confidence: 74%

“…The absence of an obvious H/D exchange effect on the decay of FAD* [1][2][3][4] indicates that FAD* is deactivated through electron transfer, as for wild-type Slr1694 (19, 21, 22). The decay of FAD* is highly multiexponential and distributed between a few picoseconds and ∼250 ps in wild-type Slr1694 (19,21,22) and the W91F mutant, which is assigned to different conformational subpopulations of the tyrosine side chain having variations in the distance to FAD, with an ensuing distribution of electron transfer rates (9,20). According to the kinetic model of Figure 4 (upper) and that for wild-type Slr1694, a single proton transfer rate of about (5 ps) -1 governs protonation of FAD •-to FADH • .…”

Section: Resultsmentioning

confidence: 99%

“…In the AppA BLUF domain it was shown that the semiconserved tryptophan in the vicinity of FAD (W104) also contributes to electron transfer to the flavin (20,37). The latter observation lended support to the "W-in" conformation of W104 in darkstate AppA.…”

mentioning

confidence: 89%

“…For the AppA BLUF domain, the observation of electron transfer from W104 to FAD (20), along with tryptophan fluorescence (25) and NMR results (9), lended support to a "W-in" conformation of W104 in dark-state AppA. The absence of such a W91-FAD electron transfer process does, however, not necessarily imply that W91 assumes the "W-out" conformation in dark-state Slr1694.…”

Section: Ref 19)mentioning

confidence: 99%

“…H 2 O. A KIE is unexpected on the FAD* lifetime because in BLUF domains electron transfer usually is considered to constitute the primary photochemical reaction (19,20,22,23,48). In addition, if proton or hydrogen transfer underlies FAD* 1 in Figure 6A deactivation, one would under most circumstances expect a slowdown of the reaction upon H/D exchange, not a speeding up.…”

Section: Ref 19)mentioning

confidence: 99%

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The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

et al. 2009

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BLUF (blue light sensing using FAD) domains belong to a novel group of blue light sensing receptor proteins found in microorganisms. We have assessed the role of specific aromatic and polar residues in the Synechocystis Slr1694 BLUF protein by investigating site-directed mutants with substitutions Y8W, W91F, and S28A. The W91F and S28A mutants formed the red-shifted signaling state upon blue light illumination, whereas in the Y8W mutant, signaling state formation was abolished. The W91F mutant shows photoactivation dynamics that involve the successive formation of FAD anionic and neutral semiquinone radicals on a picosecond time scale, followed by radical pair recombination to result in the long-lived signaling state in less than 100 ps. The photoactivation dynamics and quantum yield of signaling state formation were essentially identical to those of wild type, which indicates that only one significant light-driven electron transfer pathway is available in Slr1694, involving electron transfer from Y8 to FAD without notable contribution of W91. In the S28A mutant, the photoactivation dynamics and quantum yield of signaling state formation as well as dark recovery were essentially the same as in wild type. Thus, S28 does not play an essential role in the initial hydrogen bond switching reaction in Slr1694 beyond an influence on the absorption spectrum. In the Y8W mutant, two deactivation branches upon excitation were identified: the first involves a neutral semiquinone FADH • that was formed in ∼1 ps and recombines in 10 ps and is tentatively assigned to a FADH • -W8 • radical pair. The second deactivation branch forms FADH • in 8 ps and evolves to FAD •-in 200 ps, which recombines to the ground state in about 4 ns. In the latter branch, W8 is tentatively assigned as the FAD redox partner as well. Overall, the results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from Y8 to FAD, followed by a hydrogen bond rearrangement and radical pair recombination.Blue light photoreceptors using flavin cofactors have been the focus of recent research because of their novel mechanisms of photoactivation in contrast to "classical" photoreceptors like phytochromes and rhodopsins. Especially members of the BLUF 1 (blue light photoreceptors using FAD) family (1-3) show an especially intriguing light-induced proton network rearrangement resulting in a 10-15 nm red-shifted spectrum of the signaling state. The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded β-sheet with two R-helices packed on one side of the sheet, with the isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two R-helices (4-12). FAD is noncovalently bound to the protein through a number of hydrogen bonds and hydrophobic interactions. Figure 1 shows the three-dimensional structure of the Synechocystis Slr1694 BLUF domain (also known as PixD) in its proposed dark and light states, with the FAD binding pock...

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Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 89%

Section: Ref 19)mentioning

confidence: 99%

Section: Ref 19)mentioning

confidence: 99%

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The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

et al. 2009

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Direct Observation of Ultrafast Proton Rocking in the BLUF Domain

et al. 2022

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We present direct observation of ultrafast proton rocking in the central motif of a BLUF domain protein scaffold. The mutant design has taken consideration of modulating the proton‐coupled electron transfer (PCET) driving forces by replacing Tyr in the original motif with Trp, in order to remove the interference of a competing electron transfer pathway. Using femtosecond pump–probe spectroscopy and detailed kinetics analysis, we resolved an electron‐transfer‐coupled Grotthuss‐type forward and reverse proton rocking along the FMN–Gln–Trp proton relay chain. The rates of forward and reverse proton transfer are determined to be very close, namely 51 ps vs. 52 ps. The kinetic isotope effect (KIE) constants associated with the forward and reverse proton transfer are 3.9 and 5.3, respectively. The observation of ultrafast proton rocking is not only a crucial step towards revealing the nature of proton relay in the BLUF domain, but also provides a new paradigm of proton transfer in proteins for theoretical investigations.

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