Photocycle of the Flavin-Binding Photoreceptor AppA, a Bacterial Transcriptional Antirepressor of Photosynthesis Genes

Gauden, Magdalena; Yeremenko, Sergey; Laan, Wouter; Stokkum, Ivo H. M. van; Ihalainen, Janne A.; Grondelle, Rienk van; Hellingwerf, Klaas J.; Kennis, John T. M.

doi:10.1021/bi047359a

Cited by 175 publications

(347 citation statements)

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“…The nondecaying EADS strongly resembles the absorption of the long-lived signaling state in Slr1694 (17). We conclude that in Slr1694, the red-shifted signaling state is formed in 123 ps, consistent with previous results on the AppA BLUF domain where the long-lived product is formed on the sub-nanosecond timescale (18). Similar to AppA, FAD* in Slr1694 decays multiexponentially, albeit with substantially shorter lifetimes as judged by the decay of the stimulated emission band and the kinetic trace taken at 550 nm.…”

Section: Resultssupporting

confidence: 90%

“…This EADS evolves in 0.9 ps to the next EADS (red line), which has a lifetime of 7 ps. The 0.9-ps evolution is attributed to a vibrational cooling process of the FAD excited state (18) and involves a small blue-shift of the stimulated emission band and an increase of excited-state absorption of Ϸ510 nm, whereas the ground-state bleach remains the same. The EADS with a 7-ps lifetime thus corresponds to the vibrationally relaxed singlet excited state of FAD (hereafter denoted FAD*) and evolves to the next EADS (green line), which has a lifetime of 22 ps.…”

Section: Resultsmentioning

confidence: 96%

“…In this view, photon absorption leads to the breaking of hydrogen bonds from the Gln amino group to the N5 of flavin and to the Tyr and formation of a hydrogen bond to the O4 of the flavin. Ultrafast spectroscopy on the AppA BLUF domain showed that the red-shifted product state is formed in Ͻ1 ns, directly from the FAD singlet excited state and without any apparent reaction intermediate (18), yielding no clues on the validity of any of these scenarios.…”

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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: 90%

Section: Resultsmentioning

confidence: 96%

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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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215

435

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“…To estimate the FAD T SADS, the ground state bleach part from 420 to 500 nm was set identical to that of the singlet excited state FAD* and a low weight was assigned to the FAD T SADS between 420 and 500 nm assigned in the fitting procedure to avoid compensation effects with flavin ground state bleach present in the SADS of FAD* 1-4 , Q 1 , Q 2 , and Slr RED . The resulting FAD T SADS shows a broad, positive absorption from 500 to 690 nm, with a spectral shape that is similar to that observed in the AppA BLUF domain (35) and the LOV 1 and LOV 2 domains of phototropin (46,52,53).…”

Section: Resultssupporting

confidence: 54%

“…However, due to the instability of the long-lived photoproduct of the S28A mutant, the overall S/N ratio of this particular data set is lower than those obtained on wild-type Slr1694 and other mutants. The 0.5 ps EADS (black line) represents a mixture of the initially excited S 2 and S 1 excited states of FAD (35). After internal conversion in 0.5 ps, the 2 ps EADS (red line) represents the lowest singlet excited state of FAD, denoted as FAD*.…”

Section: Ref 19)mentioning

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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Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light‐Control in Bacteria

Baumschlager

Khammash

2021

Advanced Biology

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have distinct "dark/ground" and "lightactivated" states which confer different functions to adapt to a light stimulus.Optogenetics is a biological technique that exploits the information carrying property of light by using such photo sensors for bioengineering. Light can then be used to control genetically encoded lightsensitive proteins, which in turn can influence diverse functions of cells. The word "optogenetics" was first used in the context of light-gated ion channels in 2006. [1] The use of these channels has revolutionized neuroscience as well as other biological disciplines. The first genetically engineered light-sensitive proteins date back to 2002 when a yeast-two-hybrid system was coupled with photosensitive domains to create a light-regulated transcription system in yeast. [2] That same year, the discoveries of light-gated ion channels were published. [3,4] Since then, light has been used to perturb and control a variety of cellular functions using different approaches. These approaches, some of which will later be discussed, are either made possible through the use of light as an input, or are used as alternatives for small molecule inputs, such as chemical inducers (for gene expression) or hormones (to elicit cellular responses). In this regard, light fulfills a similar function to small molecule signals which have been used extensively in biological research and biotechnology. For example, small-molecule inducible gene expression systems are key components in synthetic biology [5] and biotechnological applications. [6] However, in contrast to small molecules, which usually bind to specific sensors, light transfers information through photons, which provides unique properties: precise spatiotemporal and orthogonal inputs.

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Photocycle of the Flavin-Binding Photoreceptor AppA, a Bacterial Transcriptional Antirepressor of Photosynthesis Genes

Cited by 175 publications

References 27 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

Synthetic Biological Approaches for Optogenetics and Tools for Transcriptional Light‐Control in Bacteria

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