On the Mechanism of Activation of the BLUF Domain of AppA

Laan, Wouter; Gauden, Magdalena; Yeremenko, Sergey; Grondelle, Rienk van; Kennis, John T. M.; Hellingwerf, Klaas J.

doi:10.1021/bi051367p

Cited by 99 publications

(148 citation statements)

References 50 publications

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“…3D). These observations are consistent with previous results on AppA, which showed no H͞D exchange effect on the excited-state lifetimes (20). FAD* has an isosbestic point at 610 nm, giving a clean view on the kinetic behavior of the intermediate species.…”

Section: Resultssupporting

confidence: 93%

“…In this way, the FAD •Ϫ and FADH • intermediates do not transiently accumulate during the reaction and therefore escape detection. A rate-limiting light-driven ET rather than a hydrogen transfer in the AppA BLUF domain is consistent with the absence of a KIE on the FAD excited-state lifetime upon H͞D exchange (20). …”

Section: Discussionsupporting

confidence: 73%

“…The potential involvement of the conserved Trp (Trp-91 in Slr1694, Trp-104 in AppA) is not taken into account because earlier work has shown that Trp-104 is not required for photocycling in AppA (20). The driving force is larger for Tyr than for Trp (net ⌬G ϭ Ϫ0.62 eV for Tyr and Ϫ0.4 eV for Trp), and (from the AppA crystal structure) the distances from Tyr-21 and Trp-104 to N5 of FAD are 4.5 and 5.7 Å, respectively (11), indicating that Tyr has a better spatial overlap with the flavin than Trp.…”

Section: Discussionmentioning

confidence: 99%

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Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Gauden

Stokkum²,

Key³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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BLUF (blue light sensing using FAD) domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae, where they control a range of physiological responses including photosynthesis gene expression, photophobia, and negative phototaxis. Other than in well known photoreceptors such as the rhodopsins and phytochromes, BLUF domains are sensitive to light through an oxidized flavin rather than an isomerizable cofactor. To understand the physicochemical basis of BLUF domain photoactivation, we have applied femtosecond transient absorption spectroscopy to the Slr1694 BLUF domain of Synechocystis PCC6803. We show that photoactivation of BLUF domains proceeds by means of a radical-pair mechanism, driven by electron and proton transfer from the protein to the flavin, resulting in the transient formation of anionic and neutral flavin radical species that finally result in the long-lived signaling state on a 100-ps timescale. A pronounced deuteration effect is observed on the lifetimes of the intermediate radical species, indicating that proton movements underlie their molecular transformations. We propose a photoactivation mechanism that involves a successive rupture of hydrogen bonds between a conserved tyrosine and glutamine by light-induced electron transfer from tyrosine to flavin and between the glutamine and flavin by subsequent protonation at flavin N5. These events allow a reorientation of the conserved glutamine, resulting in a switching of the hydrogen-bond network connecting the chromophore to the protein, followed by radicalpair recombination, which locks the glutamine in place. It is suggested that the redox potential of flavin generally defines the light sensitivity of flavin-binding photoreceptors.flavoprotein ͉ light sensing ͉ photochemistry ͉ reaction mechanism ͉ spectroscopy

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Section: Resultssupporting

confidence: 93%

Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Gauden

Stokkum²,

Key³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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217

435

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“…This observation is quite remarkable given that in the AppA BLUF domain the corresponding mutation leads to acceleration of the photocycle (37). The S28A mutant has a dark recovery of 4 s, essentially the same as in WT.…”

Section: Resultsmentioning

confidence: 77%

“…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%

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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Influence of a Joining Helix on the BLUF Domain of the YcgF Photoreceptor from Escherichia coli

et al. 2008

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BLUF-domain-comprising photoreceptors sense blue light by utilizing FAD as a chromophore. The ycgF gene product of Escherichia coli is composed of a N-terminal BLUF domain and a C-terminal EAL domain, with the latter postulated to catalyze c-di-GMP hydrolysis. The linkage between these two domains involves a predominantly helical segment. Its role on the function of the YcgF photoreceptor domain was examined by characterizing BLUF domains with and without this segment and reconstituting them with either FAD, FMN or riboflavin. The stability of the light-adapted state of the YcgF BLUF domain depends on the presence of this joining, helical segment and the adenosine diphosphate moiety of FAD. In contrast to other BLUF domains, two-dimensional (1)H,(15)N and one-dimensional (1)H NMR spectra of isotope-labeled YcgF-(1-137) revealed large conformational changes during reversion from the light- to the dark-adapted state. Based on these results the function of the joining helix in YcgF during signal transfer and the role of the BLUF domain in regulating c-di-GMP levels is discussed.

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On the Mechanism of Activation of the BLUF Domain of AppA

Cited by 99 publications

References 50 publications

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

Hydrogen-bond switching through a radical pair mechanism in a flavin-binding photoreceptor

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

Influence of a Joining Helix on the BLUF Domain of the YcgF Photoreceptor from Escherichia coli

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