Excited State Absorption Spectra

RF/DMSO:The evolution of the RF excited state absorption spectrum upon excitation at 400 nm is shown in Figure 6.24, left, up to 30 ps; decomposition of the transient absorption was accomplished with the procedure outlined in Section 6.5.5. After a small initial blue shift of the 500 nm band, the spectrum stays unchanged from 1.5 to 30 ps, demonstrating that the population remains in the excited state.

FAD/H₂O:The evolution of the excited state absorption of FAD/H₂O upon excitation at 440 nm is shown in Figure 6.24, right, up to 30 ps. On the longer time scale the adenine-induced decay is apparent. This is accompanied by a small change in the shape of the 516 nm ESA band; otherwise the spectrum decays uniformly.

6.7.4 Femtosecond Stimulated Resonance Raman Spectroscopy

Transient stimulated Raman spectra of FAD/H₂O and RF/DMSO with Raman wave-lengths of 500 nm and 523 nm (only FAD) are compared in Figure 6.25. The spectral

6 Excited Flavin: A Femtosecond Stimulated Raman Study

Figure 6.24: Excited-state absorption spectra of RF/DMSO excited at 400 nm (left) and FAD in water excited at 440 nm (right) under magic-angle conditions. The spectra are the result of a decomposition of the transient-absorption spectra as described in Section 6.5.5.

shape was already described before. It is similar for all measurements with visible Ra-man pulses, and is dominated by negative features at the positions of the ground-state bands. At longer delay times the spectra decay according to the population dynam-ics, which were observed already in transient absorption and fluorescence upconversion.

Within the first picoseconds additional decay is seen, which probably originates from population depletion by the Raman pulse (see Figure 6.10 and the associated discus-sion). The spectral shape stays similar throughout the evolution although the relative intensities may exhibit minor changes. On account of the complicated signal I refrain from a deeper discussion.

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6.7 Supporting Material

Figure 6.25: Transient stimulated resonance Raman spectra of FAD/H₂O (left) and RF in DMSO (right) recorded with 500 nm and 523 nm Raman pulses. The sample was actinically excited at 475 nm; polarization conditions are indicated as || (parallel) and ma (magic angle). Transient spectra at different time scales were acquired in separate measurements in time steps of 6 fs, 100 or 150 fs, and 3 or 6 ps, respectively. For presentation, the transient evolution with 500 nm Raman excitation was smoothed by a 5 point moving-average in time.

7 Light-Induced Changes in BLUF Photoreceptors

7.1 Introduction

Among the various flavoproteins, photoreceptors that contain BLUF (Blue Light Using FAD) domains have attracted considerable attention in recent years.[83,168,233–235] This is because upon blue-light excitation, the signaling state is formed already within several hundreds of picoseconds. Spectroscopically only a small (10–15 nm) shift of the absorp-tion spectrum is observed (Figure 7.1), accompanied by subtle changes in vibraabsorp-tional spectra.[161–163,171,236] Surprisingly, the formed light-adapted state which carries these spectra is stable for seconds to minutes.

The biological function of such receptors is highly diverse and still not completely un-derstood. It is known that the protein AppA from the phototropic bacteriaRhodobacter sphaeroides regulates the photosynthetic gene expression.^[237,238] Another example for this class of receptors are the PAC (photoactivated adenyl cyclase) proteins from the

flag-Figure 7.1:Dark- and light-adapted state of the wild-type BLUF photoreceptor Slr1694 (top), and light-minus-dark difference spectrum (bottom). The spectra were kindly provided by Tilo Mathes (see footnote on page 161 for contact details).

7 Light-Induced Changes in BLUF Photoreceptors

Figure 7.2: The wild-type BlrB photoreceptor. Left: holoprotein. Middle and right:

chromophore binding site with the residues around the isoalloxazine ring and leucine 66.

Structure from the RCSB protein database, ref. 241.

ellaEuglena gracilis, which are responsible for a photophobic reaction upon blue-light illumination.^[239] Recently also a photoactivated adenyl cyclase from the bacterium Beg-giatoa was characterized. Another protein, BlrP1 from Klebsiella pneumoniae controls motility, virulence and antibiotic resistence in the bacteria.^[240]

The protein structure of several BLUF domains was solved by X-ray crystallogra-phy.[162,171,240–244] Figures 7.2 and 7.3 show the structures of the holoprotein and the flavin binding site for BlrB fromRhodobacter sphaeroidesand Slr1694 from the cyanobac-terium Synechocystis sp. PCC6803.^[171,241] These short proteins consist mainly of the BLUF domain itself with a C-terminal extension of unknown function.^[171,241] The ter-tiary structure shows a ferredoxin-like fold, consisting of a five-stranded mixed β-sheet with two parallelα-helices on one side and a helix-turn-helix motif on the other. The flavin is bound noncovalently to two of theα-helices, while the ribityl chain extends to the surface of the protein and the adenosine diphosphate moiety is exposed to the sol-vent. The adenine is not crucial for the formation of the light-adapted state,^[163,241]but it may serve as a recognition site for effector domains of other proteins.^[241] The binding site of the isoalloxazine ring is heterogeneous: whereas the dimethyl benzene ring is in a non-polar environment, the pyrimidine part interacts with polar residues and forms several hydrogen bonds with the protein backbone. Involvement in the formation of the signaling state was discussed for the conserved amino acids tyrosin-8, glutamine-50, and the semi-conserved tryptophan-91 (from here on, the notation for Slr1694 is adopted).

The arrangement of the binding site in the dark- and light-adapted state is subject to controversial discussion. For glutamine-50 different orientations were suggested by X-ray and NMR experiments:^[243–245] the amino group may be either either hydrogen-bonded to tyrosine-8, or else to the FAD C(4)=O. On this basis it was suggested that

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7.1 Introduction

Figure 7.3: The wild-type Slr1694 photoreceptor. Left: holoprotein. Right: two differ-ent conformations of the chromophore binding site. The structure was taken from the RCSB protein database, ref. 171.

signaling state formation involves a ∼ 180^◦ rotation of the conserved glutamine with a concomitant change of the hydrogen-bond network.[165,243,244] This agrees with re-sults from infrared- and Raman-difference spectroscopy, which found a weakening of the C(4)=O and C(2)=O bonds and a strengthening of the N(1)C(10a) and/or C(4a)N(5) bonds upon formation of the light-adapted state.[161,162,236] Mutation of the glutamine abolishes the ability of the protein to undergo the photocycle.^[246]

Tyrosine-8 is another conserved amino acid that is crucial for the formation of the sig-naling state.[166,236,242,246–249] Early NMR studies on the AppA BLUF domain suggested that an increase of π stacking between the isoalloxazine ring and the conserved tyrosine is responsible for the absorption red-shift.^[248]The geometry of the binding site, however, is unfavorable for such interaction,^[241,244] and more recent publications explain the role of the tyrosine by its influence on the orientation of glutamine-50 and its ability to act as an electron donor for an intermediate reduction of the flavin chromophore.[165,166,246]

Tryptophan-91 is found in the majority of BLUF domains, but it not essential for signaling state formation. Mutatants, in which Trp-91 is replaced by alanine or pheny-lalanine, still show the characteristic∼10 nm red-shift of the absorption spectrum upon illumination, but with a slightly increased rate for the dark-state recovery.^[171,246]As an aromatic amino acid, tryptophan-91 can act as an alternative electron donor for a light-induced reduction of flavin.^[166,246] In crystal structures, tryptophan-91 was found in different orientations, see Figure 7.3 (middle and right).^[171] It adopted either a solvent-exposed conformation (conformation A), or was buried inside the protein and in contact with FAD (conformation B). The orientation of tryptophan-91 is connected to the posi-tion of Methionin-93, which is highly conserved.^[249] It was suggested that tryptophan-91 changes its orientation upon formation of the signaling state and acts as a switch that couples the initial glutamine motion to the protein backbone.[166,171,243,250]

7 Light-Induced Changes in BLUF Photoreceptors

Amino acid residues around the ribityl and phosphate chain are not essential for the photocycle. A mutation of leucine-66 in BlrB to phenylalanine (see Figure 7.2), for example, does not impede the formation of the signaling state.^[249] Nonetheless, subtle changes of the protein backbone upon mutation lead to a 6× faster return to the dark-adapted form, already within 1.3 s.^[249] In this work, the acceleration of dark-state recovery for BlrB-L66F is utilized to avoid accumulation of the light-adapted dark-state during transient absorption measurements. Femtosecond absorption spectroscopy is only sensitive to fast changes around the isoalloxazine ring, so that the measured evolution should not observe effects from the the L66 mutation. The discussed leucine is not conserved among the BLUF domains of different proteins; in Slr1694, for instance, an arginine is found in this position (Arg-65, see Figure 7.3).

The ultrafast evolution upon excitation has been studied for several BLUF photore-ceptors by transient absorption, fluorescence, and infrared spectroscopy,[165–169,246,251,252]

leading to the following idea about the formation of the BLUF signaling state: The ex-cited chromophore is reduced by electron transfer from the conserved tyrosine within several picoseconds. The generated flavin radical anion (FAD^−•) is then protonated by tyrosine-8 to the neutral flavin radical (FADH^•). The concomitant changes in the charge distribution may induce a rotation of glutamine-50. Subsequent hydrogen back-transfer on the 100 ps time scale recovers the oxidized flavin, but embedded into a modified hydrogen-bond network with an ∼ 180^◦ rotated glutamine. Evidence for the radical intermediates was found for the slr1694 protein^[165,167]and several mutants of BLUF do-mains,^[166,246] but they did not accumulate in transient absorption experiments on the AppA protein.^[251] A general observation in transient experiments on BLUF domains is the multi-exponential character of the measured dynamics, which has been assigned to a conformational heterogeneity of the chromophore binding site.

The interpretation of previous transient absorption measurements was hampered by two limitations: (i) the sample was excited at 400 nm, thereby populating a mixture of S₁ and the S₂ states. By pumping specifically the S₁ ←S₀ transition, one should be able to reduce the complexity of the measured dynamics. (ii) The spectral window for probing was limited to wavelengths > 400 nm, so that generated intermediates were identified solely by their absorption in the visible, overlapping at early delay times with the stimulated emission. To increase the available information, it is desirable to extend the spectral range to the UV region.

In this work, broadband transient absorption is applied to investigate light-induced changes in the BlrB-L66F mutant over a spectral range from 330 to 1000 nm. Informa-tion on the role of the tryptophane-91 as a potential electron donor is obtained from measurements on the Slr1694-Y8F mutant. Selective excitation is assured by using pump pulses at 440–480 nm.

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7.2 Experimental Details

7.2 Experimental Details

7.2.1 Transient Absorption

The transient spectral evolution, presented here, is the average of several measurement runs. Within precision, the transient absorption did not change between consecutive measurements, and product accumulation was not observed.

Transient absorption spectra of BlrB-L66F were recorded with the pump-supercon-tinuum probe (PSCP) setup based on theCLARK MXRCPA 2001 laser system. The photoreceptor was excited with 30 fs NOPA pulses at 480 nm (0.5 µJ). Measurements were carried out under parallel polarization conditions. From NMR spectroscopy a rotational correlation time of 8.2 ns was deduced for the monomeric protein,^[249] so that the effect of rotational diffusion is considered to be negligible for the present experiment.

Replacement of the sample after each shot was assured by circulating the sample through a flow cell. For the presentation in Figure 7.5, spectra up to 5 ps and > 50 ps were smoothed by 50 fs and 11 ps moving averages in time, respectively.

Transient absorption spectra of Slr1694-Y8F were recorded with the pump-supercon-tinuum probe setup based on the Femtolasers sPro laser system. Pump pulses at 440 nm (50 fs fwhm, 0.5 µJ) were obtained by mixing the 800 nm fundamental with the frequency-doubled output of the TOPAS parametric amplifier. Measurements were carried under parallel and perpendicular polarization conditions, and the magic angle signal was calculated as ∆OD_ma = ∆OD_||+ 2∆OD⊥

/3. The sample was replaced between consecutive laser shots by laterally moving the sample cell by ∼0.5 cm with an oscillation frequency of 10 Hz, and slowly varying the vertical position of the cell.