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.

The transcriptional antirepressor AppA from the photosynthetic bacterium Rhodobacter sphaeroides senses both the light climate and the intracellular redox state. Under aerobic conditions in the dark, AppA binds to and thereby blocks the function of PpsR, a transcriptional repressor. Absorption of a blue photon dissociates AppA from PpsR and allows the latter to repress photosynthesis gene expression. The N terminus of AppA contains sequence homology to flavin‐containing photoreceptors that belong to the BLUF family. Structural and chemical aspects of signal transduction mediated by AppA are still largely unknown. Here we present NMR studies of the N‐terminal flavin‐binding BLUF domain of AppA. Its solution structure adopts an α/β‐sandwich fold with a βαββαββ topology, which represents a new flavin‐binding fold. Considerable disorder is observed for residues near the chromophore due to conformational exchange. This disorder is observed both in the dark and in the light‐induced signaling state of AppA. Furthermore, we detect light‐induced structural changes in a patch of surface residues that provide a structural link between light absorption and signal‐transduction events.

ArcBA is a two-component regulatory system of Escherichia coli involved in sensing oxygen availability and the concomitant transcriptional regulation of oxidative and fermentative catabolism. Based on in vitro data, it has been postulated that the redox state of the ubiquinone pool is the determinant for ArcB kinase activity. Here we report on the in vivo regulation of ArcB activation, as determined using a lacZ reporter specifically responsive to phosphorylated ArcA. Our results indicate that upon deletion of a ubiquinone biosynthetic enzyme, regulation of ArcB in the anaerobic-aerobic transition is not affected. In contrast, interference with menaquinone biosynthesis leads to inactivation of ArcB during anaerobic growth; this phenotype is fully rescued by addition of a menaquinone precursor. This clearly demonstrates that the menaquinones play a major role in ArcB activation. ArcB shows a complex pattern of regulation when E. coli is titrated through the entire aerobiosis range; ArcB is activated under anaerobic and subaerobic conditions and is much less active under fully aerobic and microaerobic conditions. Furthermore, there is no correlation between ArcB activation and the redox state of the ubiquinone pool, but there is a restricted correlation between the total cellular ubiquinone content and ArcB activity due to the considerable increase in the size of the ubiquinone pool with increasing degrees of aerobiosis. These results lead to the working hypothesis that the in vivo activity of ArcB in E. coli is modulated by the redox state of the menaquinone pool and that the ubiquinone/ubiquinol ratio in vivo surely is not the only determinant of ArcB activity.

The flavin adenine dinucleotide (FAD)-containing photo-receptor protein AppA (in which the FAD is bound to a novel so-called BLUF domain) from the purple nonsulfur bacterium Rhodobacter sphaeroides was previously shown to be photo-active by the formation of a slightly redshifted long-lived intermediate that is thought to be the signaling state. In this study, we provide further characterization of the primary photochemistry of this photoreceptor protein using UV-Vis and Fourier-transform infrared spectroscopy, pH measurements and site-directed mutagenesis. Available evidence indicates that the FAD chromophore of AppA may be protonated in the receptor state, and that it becomes exposed to solvent in the signaling state. Furthermore, experimental data lead to the suggestion that intramolecular proton transfer (that may involve [anionic] Tyr-17) forms the basis for the stabilization of the signaling state.

BLUF (blue-light sensing using FAD) domain proteins are a novel group of blue-light sensing receptors found in many microorganisms. The role of the aromatic side chains Y21 and W104, which are in close vicinity to the FAD cofactor in the AppA BLUF domain from Rhodobacter sphaeroides, is investigated through the introduction of several amino acid substitutions at these positions. NMR spectroscopy indicated that in the W104F mutant, the local structure of the FAD binding pocket was not significantly perturbed as compared to that of the wild type. Time-resolved fluorescence and absorption spectroscopy was applied to explore the role of Y21 and W104 in AppA BLUF photochemistry. In the Y21 mutants, FADH • -W • radical pairs are transiently formed on a ps time scale and recombine to the ground state on a ns time scale. The W104F mutant shows a spectral evolution similar to that of wild type AppA but with an increased yield of signaling state formation. In the Y21F/W104F double mutant, all light-driven electron-transfer processes are abolished, and the FAD singlet excited-state evolves by intersystem crossing to the triplet state. Our results indicate that two competing light-driven electrontransfer pathways are available in BLUF domains: one productive pathway that involves electron transfer from the tyrosine, which leads to signaling state formation, and one nonproductive electron-transfer pathway from the tryptophan, which leads to deactivation and the effective lowering of the quantum yield of the signaling state formation. Our results are consistent with a photoactivation mechanism for BLUF domains where signaling state formation proceeds via light-driven electron and proton transfer from the conserved tyrosine to FAD, followed by a hydrogen-bond rearrangement and radical-pair recombination.

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.

Many bioinspired transition-metal catalysts have been developed over the recent years. In this review the progress in the design and application of ligand systems based on peptides and DNA and the development of artificial metalloenzymes are reviewed with a particular emphasis on the combination of phosphane ligands with powerful molecular recognition and shape selectivity of biomolecules. The various approaches for the assembly of these catalytic systems will be highlighted, and the possibilities that the use of the building blocks of Nature provide for catalyst optimisation strategies are discussed.

Upon heterologous expression of the BLUF (for: Blue-Light sensing Using Flavin) domain from AppA, a transcriptional anti-repressor from Rhodobacter sphaeroides, in Escherichia coli, photoactive holo-protein is formed through non-covalent binding of a flavin. Whereas it is generally assumed that FAD is the physiological chromophore of this photo-perception domain in vivo, E. coli can (and does) insert, depending on the growth conditions, all naturally occurring flavins, i.e. riboflavin, FMN and FAD into this protein domain. The nature of the particular flavin bound affects the photochemical- and particularly the fluorescence properties of the N-terminal domain of this photosensory protein.