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

Gauden, Magdalena; Stokkum, Ivo H. M. van; Key, Jason; Lührs, Daniel C.; Grondelle, Rienk van; Hegemann, Peter; Kennis, John T. M.

doi:10.1073/pnas.0600720103

Cited by 216 publications

(484 citation statements)

References 39 publications

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“…It shows further decay of ground state bleach and stimulated emission features and a decay of the transient band at 600 nm. A prominent positive band has come up near 490 nm which is indicative of long-lived product (BLUF RED , also referred to as Slr RED in the specific case of Slr1694) formation (19). 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.…”

Section: Resultsmentioning

confidence: 99%

“…The first EADS evolves to the second EADS in 1.4 ps and involves minor spectral changes that include a loss of bleach near 470 nm and a slight blue shift of the stimulated emission band near 550 nm. This evolution can be assigned to a vibrational cooling process on the excited FAD chromophore in combination with excited-state decay (19,35,47).…”

Section: Resultsmentioning

confidence: 99%

“…Ultrafast Spectroscopy of Wild-Type Slr1694. Ultrafast transient absorption spectroscopy on wild-type Slr1694 was performed previously in our laboratory (19). The ultrafast experiments reported in this paper were performed on a different laser setup (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Mutations were introduced into pET28a(þ)-slr1694 (19,42) by site-directed mutagenesis (QuikChange; Stratagene) using the primers in Table 1. The mutated DNA was confirmed by sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The current opinion on the photoactivation of the BLUF domains as observed by steady-state FTIR (14)(15)(16), Raman (17,18), ultrafast visible and IR spectroscopy (19)(20)(21)(22)(23)(24)(25), and quantumchemical calculations (26)(27)(28)(29) includes light-induced radical pair formation by electron and proton transfer from an essential tyrosine side chain (Y8) to the flavin cofactor on the picosecond time scale. Subsequently, hydrogen bond rearrangement takes place, possibly involving ∼180°rotation of the amide group of an essential glutamine side chain (Q50) which is followed by radical pair recombination in less than 100 ps.…”

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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: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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Changes in Active Site Hydrogen Bonding upon Formation of the Electronically Excited State of Photoactive Yellow Protein

Hoff

Kang

Kumauchi

et al. 2010

Hydrogen Bonding and Transfer in the Excited State

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Light plays three main roles in biology. First, light from the sun provides the main source of energy driving the biosphere through chlorophyll-based electron transfer in photosynthetic reaction centers and retinal-based proton pumping in rhodopsins [1]. Second, light is a source of information about the environment for many organisms using photosensory proteins, including animals, plants, fungi and bacteria [2,3]. Third, light can cause damage to biological systems. Light from the near-UV region can damage DNA [4], and excess visible light can damage the photosynthetic machinery in photoinhibition [5]. Thus, light is of great importance for a wide range of biological processes.Proteins that perform photosynthesis or light sensing consist of an apoprotein and a bound chromophore. Only a small number of chromophores account for most known processes in photobiology: chlorophyll and linear tetrapyrroles, caretoids and retinal, flavins and p-coumaric acid [2]. In many cases the chromophore has strong and functionally important interactions with the protein binding pocket, particularly charge-charge interactions and hydrogen bonding interactions. While many different proteins interact with light in a wide range of organisms, almost all of these systems are based on key types of photochemical processes, including electron transfer, C¼C double bond isomerization and the formation of chemical bonds [2,6]. These photochemical events often trigger a cascade of thermal reactions in the protein that extends to the millisecond and minute range, and that result in a biologically relevant output. If the cascade of thermal reactions results in the re-formation of the initial state, the process is referred to as a photocycle. It is the initial ultrafast

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

Cited by 216 publications

References 39 publications

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

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

Changes in Active Site Hydrogen Bonding upon Formation of the Electronically Excited State of Photoactive Yellow Protein

Direct Observation of Ultrafast Proton Rocking in the BLUF Domain

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