The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal domain belonging to a new class of photoreceptors designated BLUF domains. AppA was shown to control photosynthesis gene expression in response to blue light and oxygen tension. We have investigated the photocycle of the AppA BLUF domain by ultrafast fluorescence, femtosecond transient absorption, and nanosecond flash-photolysis spectroscopy. Time-resolved fluorescence experiments revealed four components of flavin adenine dinucleotide (FAD) excited-state decay, with lifetimes of 25 ps, 150 ps, 670 ps, and 3.8 ns. Ultrafast transient absorption spectroscopy revealed rapid internal conversion and vibrational cooling processes on excited FAD with time constants of 250 fs and 1.2 ps, and a multiexponential decay with effective time constants of 90 ps, 590 ps, and 2.7 ns. Concomitant with the decay of excited FAD, the rise of a species with a narrow absorption difference band near 495 nm was detected which spectrally resembles the long-living signaling state of AppA. Consistent with these results, the nanosecond flash-photolysis measurements indicated that formation of the signaling state was complete within the time resolution of 10 ns. No further changes were detected up to 15 micros. The quantum yield of the signaling-state formation was determined to be 24%. Thus, the signaling state of the AppA BLUF domain is formed on the ultrafast time scale directly from the FAD singlet excited state, without any apparent intermediate, and remains stable over 12 decades of time. In parallel with the signaling state, the FAD triplet state is formed from the FAD singlet excited state at 9% efficiency as a side reaction of the AppA photocycle.

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AppA, a transcriptional antirepressor, regulates the steady expression of photosynthesis genes in Rhodobacter sphaeroides in response to high-intensity blue light and to redox signals. Its blue-light sensing is mediated by an N-terminal BLUF domain, a member of a novel flavin fold. The photocycle of this domain (AppA(5-125)) includes formation of a slightly red-shifted long-lived signaling state, which is formed directly from the singlet excited state of the flavin on a subnanosecond time scale [Gauden et al. (2005) Biochemistry 44, 3653-3662]. The red shift of the absorption spectrum of this signaling state has been attributed to a rearrangement of its hydrogen-bonding interactions with the surrounding apoprotein. In this study we have characterized an AppA mutant with an altered aromatic amino acid: W104F. This mutant exhibits an increased lifetime of the singlet excited state of the flavin chromophore. Most strikingly, however, it shows a 1.5-fold increase in its quantum yield of signaling state formation. In addition, it shows a slightly increased rate of ground-state recovery. On top of this, the presence of imidazole, both in this mutant protein and in the wild-type BLUF domain, significantly accelerates the rate of ground-state recovery, suggesting that this rate is limited by rearrangement of (a) hydrogen bond(s). In total, an approximately 700-fold increase in recovery rate has been obtained, which makes the W104F BLUF domain of AppA, for example, suitable for future analyses with step-scan FTIR. The rate of ground-state recovery of the BLUF domain of AppA follows Arrhenius kinetics. This suggests that this domain itself does not undergo large structural changes upon illumination and that the structural transitions in full-length AppA are dominated by interdomain rearrangements.

We present a study of the effect of hydrogen bonding on vibrational energy relaxation of the OH-stretching mode in pure water and in water-acetonitrile mixtures. The extent of hydrogen bonding is controlled by dissolving water at various concentrations in acetonitrile. Infrared frequency-resolved pump-probe measurements were used to determine the relative abundance of hydrogen-bonded versus non-hydrogen-bonded OH bonds in water-acetonitrile mixtures. Our data show that the main pathway for vibrational relaxation of the OH-stretching mode in pure water involves the overtone of the bending mode. Hydrogen bonding is found to accelerate the population relaxation from 3 ps in dilute solutions to 700 fs in neat water, as a result of increasing overlap between donor and acceptor modes. Hydroxyl groups that initially are not hydrogen bonded have two relaxation pathways: by direct nonresonant relaxation to the bending mode with a time constant of 12 ps or by making a hydrogen bond to a neighboring water molecule first (∼2 ps) and then relaxing as a hydrogen-bonded OH oscillator.

Time-resolved ultraviolet-visible spectroscopy was used to characterize the photocycle transitions in single crystals of wild-type and the E-46Q mutant of photoactive yellow protein (PYP) with microsecond time resolution. The results were compared with the results of similar measurements on aqueous solutions of these two variants of PYP, with and without the components present in the mother liquor of crystals. The experimental data were analyzed with global and target analysis. Distinct differences in the reaction path of a PYP molecule are observed between these conditions when it progresses through its photocycle. In the crystalline state i), much faster relaxation of the late blue-shifted photocycle intermediate back to the ground state is observed; ii), this intermediate in crystalline PYP absorbs at 380 nm, rather than at 350-360 nm in solution; and iii), for various intermediates of this photocycle the forward reaction through the photocycle directly competes with a branching reaction that leads directly to the ground state. Significantly, with these altered characteristics, the spectroscopic data on PYP are fully consistent with the structural data obtained for this photoreceptor protein with time-resolved x-ray diffraction analysis, particularly for wild-type PYP.

Femtosecond two-color vibrational pump−probe spectroscopy is used to investigate the interaction between the NH- and CO-stretch vibrations in a rotaxane composed of a benzylic amide macrocycle mechanically locked onto a succinamide-based thread. From the transient absorption spectrum, we obtain the cross anharmonicities and cross-peak anisotropies arising from the NH(macrocycle)/CO(macrocycle) and NH(macrocycle)/CO(thread) interactions. The cross-peak anisotropies are used to determine angles between NH and CO bonds in the macrocycle and the thread, providing structural information with picosecond temporal resolution. The CO and NH groups that form the macrocycle−thread hydrogen bonds are found to interact much more strongly than the CO and NH groups in other molecular systems containing the same NH···OC hydrogen-bond motif. We attribute this enhancement of the NH/CO anharmonic interaction to a cooperative effect, by which the two ring−thread hydrogen bonds sharing a hydrogen-bond acceptor mutually amplify each other. The relaxation dynamics of the NH/CO cross peaks has also been investigated. Surprisingly, the NH/CO cross peak observed upon exciting the NH-stretch mode decays much more slowly than the corresponding diagonal NH-stretch peak. This can be explained by the presence of an intermediate state that becomes populated in the NH-stretch vibrational relaxation and that is coupled to the CO-stretch mode. Our results demonstrate that NH/CO heterovibrational 2D-IR spectroscopy is well suited to observe the elementary hydrogen-bond making and breaking steps involved in the motion of rotaxane-based molecular devices.

Heterodyne-detected transient grating experiments on the OH-stretch mode of HDO dissolved in D 2 O resolve two distinctly different contributions originating from the initially excited OH stretch and the OD stretch which is thermally activated during the OH population relaxation. It is demonstrated that interference of both contributions greatly affects the outcome of IR photon echo experiments.Water, being a relatively simple substance in the form of a single, isolated molecule, possesses intricate and even peculiar properties in the liquid phase ͓1͔. The complexity of liquid water is largely determined by a three-dimensional ͑3D͒ network of hydrogen bonds, which is in constant movement on ultrafast time scales. Recent years have witnessed an impressive progress in studying the dynamical processes in water due to considerable advances in IR femtosecond spectroscopy ͓2,3͔. Especially different photon echo ͑PE͒ techniques have been proven exceptionally useful in unraveling the water dynamics at subpicosecond time scales ͓4-13͔. These experimental and theoretical studies are mainly focused on the OH or OD stretching vibrations of an HDO molecule embedded in D 2 O or H 2 O, respectively. In this manner, a number of unwanted complications such as excitation delocalization or Förster energy transfer ͓14͔ is circumvented. The normal or heavy water around the HDO molecule is deemed as a dynamical bath in a similar fashion as outlined by the Multimode Brownian Oscillator ͑MBO͒ model for electronic transitions ͓15͔ while the probe OH / OD stretching vibration is considered to be "the system." The bath modulates the vibrational frequency of the system and can in turn react to the state of the system ͑i.e., excited vibration͒ to account for the vibrational Stokes shift ͓16͔.The OH / OD molecular stretching vibration is incredibly sensitive to the strength of the hydrogen bond network ͓3,17,18͔. The picosecond stretching mode lifetime provides a fast and efficient energy transfer from the pump-excited OH / OD oscillators to low-frequency thermal modes raising temperature in the focal volume. As a result, the hydrogenbond network weakens or even breaks ͓19͔ which changes the dynamical bath around the probe HDO molecule ͓20-22͔. Consequently, the OH / OD stretch absorption of the probe HDO molecule decreases and its spectrum shifts toward higher frequencies ͓23͔. The backreaction of the bath to the system has been successfully incorporated into the MBO model to account for a number of experimental features observed in pump-probe spectroscopy ͓5,11,19͔. However, attempts to straightforwardly transfer this approach to the transient grating ͑TG͒ and PE echo spectroscopy required an

To properly respond to changes in fluency conditions, Nature has developed a variety of photosensors that modulate gene expression, enzyme activity and/or motility. Dedicated types have evolved, which can be classified in six families: rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins and BLUF-proteins. The photochemistry of the first three families is based on cis/trans isomerization of an ethylene bond. Surprisingly, the latter three all use flavin as their chromophore, but each with very different photochemistry. In this contribution we will discuss the molecular basis of signal generation in a xanthopsin (Photoactive Yellow Protein (PYP) from Halorhodospira halophila), a photoreceptor for negative phototaxis, and in a BLUF protein (AppA from Rhodobacter sphaeroides), a transcriptional anti-repressor. PYP is activated through trans/cis isomerization of the 7,8-vinyl bond of its 4-hydroxycinnamic acid chromophore. This initiates a photocycle with multiple intermediates, like pB, which is formed after intramolecular proton transfer. The negative charge thus formed in the interior of the protein triggers formation of a partially unfolded signaling state. For AppA much less is known about the underlying photochemistry. Available evidence suggests that it is based on a light-induced change in the hydrogen-bonding of its flavin chromophore and/or a change in hydrophobic stacking between the flavin and/or nearby aromatic amino acids like Y 21. A signaling state is formed within microseconds, which recovers with a rate of approximately 10(-3) s(-1). The change in conformation between receptor- and signaling-state in AppA, however, appear to be minute as compared to those in PYP. Here we review the underlying chemistry in the various steps of the photocycle of these two photoreceptor proteins and provide new data on their mechanism and function.