The Solution Structure of the AppA BLUF Domain: Insight into the Mechanism of Light‐Induced Signaling

Grinstead, Jeffrey S.; Hsu, Shang-Te Danny; Laan, Wouter; Bonvin, Alexandre M. J. J.; Hellingwerf, Klaas J.; Boelens, Rolf; Kaptein, Robert

doi:10.1002/cbic.200500270

Cited by 121 publications

(225 citation statements)

References 39 publications

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“…6 and 7 and Supporting Text, which are published as supporting information on the PNAS web site, along with a detailed description of the applied assumptions and premises. In short, four lifetimes are assumed for the decay of FAD*, which stem from inhomogeneity in the groundstate population as shown for the AppA BLUF domain (14,18). FAD* evolves to a first intermediate Q 1 , which in turn evolves to a second intermediate Q 2 .…”

Section: Resultsmentioning

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.

217

435

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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: Resultsmentioning

confidence: 99%

“…Recently, crystal and solution structures of several BLUF domains were obtained (11)(12)(13)(14). The BLUF domain shows a ferredoxin-like fold consisting of a five-stranded ␤-sheet with two ␣-helices packed on one side of the sheet, with the noncovalently bound isoalloxazine ring of flavin adenine dinucleotide (FAD) positioned between the two ␣-helices.…”

mentioning

confidence: 99%

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.

217

435

View full text Add to dashboard Cite

show abstract

“…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%

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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“…In various x-ray crystal and NMR solution structures (14)(15)(16)(17)(18)28), as well as spectroscopic (13,22,(39)(40)(41) and theoretical (42)(43)(44) studies, the dark-state orientation of Gln-63 has remained ambiguous. As illustrated in Fig.…”

Section: Structure Of the Bluf Domain Of Appamentioning

confidence: 99%

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

Unno¹,

Kikuchi²,

Masuda³

et al. 2010

AIP Conference Proceedings

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The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp in conformation) or exposed to the solvent (Trp out conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp in conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

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The Solution Structure of the AppA BLUF Domain: Insight into the Mechanism of Light‐Induced Signaling

Cited by 121 publications

References 39 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

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

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