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

Abstract

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...

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

“…It remains unclear how the imidic tautomer is maintained for any length of time, given the roughly 40 kJ/mol higher energy (36), but the stronger hydrogen bonds formed by Gln-48 with Tyr-6 and the flavin must offset the energy cost to some extent, as will the stronger Asn-30 hydrogen bonds to the chromophore. Different BLUF proteins show different behavior if the conserved tryptophan is replaced with phenylalanine or alanine, with recovery of the dark state being accelerated in some cases and retarded in others, but dark-state activity is increased (23,37,38). A similar pseudolit state is found if the key methionine is replaced with alanine, which can also be explained by a change in the relative stability of different orientations of the tryptophan sidechain at the protein surface (16,38).…”

“…FTIR showed that light exposure weakens the carbonyl bonds at C2 and C4 of the flavin, but suggested much larger changes in the protein itself (21,22). Stronger hydrogen bonding between the flavin O4 atom and the neighboring glutamine residue can explain the spectral shifts (13,(21)(22)(23) and is reflected in changes at the exterior of the BLUF domain, near the last β-strand and the surface tryptophan. This change leads to different downstream effects in different proteins; for example in BlrP1, it affects the coordination of essential metal ions at the active site of the neighboring domain (7).…”

Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors

“…Mutants and Protein Production-Slr1694 mutants were produced from pET28(ϩ)-slr1694, as described previously (44). Site-directed mutations were introduced according to the QuikChange TM (Stratagene) protocol using the primer pairs N31R/N31R_r and N31H/N31H_r as indicated (Table 2).…”

“…CpX⌬Y cells transformed with pET28a(ϩ)-slr1694 were grown Ultrafast Transient Absorption Spectroscopy and Data Analysis-Visible absorption spectroscopy was carried out using pump-probe setups as described previously (44,52). The reaction was induced at 400 nm with an energy of ϳ800 nJ/pulse.…”

Transient conformational fluctuation of TePixD during a reaction

“…The absorption spectrum does not change further, and the dark state is recovered with a time constant of ∼5 s at room temperature (40,43). The spectral red shift was explained by the rearrangement of the hydrogen bond network around the chromophore (6,(46)(47)(48). The TG method has revealed the dynamic photoreaction mechanism, which cannot be detected by conventional spectroscopic methods.…”