Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin.

The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.

FTIR Spectroscopy Revealing Light-Dependent Refolding of the Conserved Tongue Region of Bacteriophytochrome.

Bacteriophytochromes (BphPs) constitute a class of photosensory proteins that toggle between Pr and Pfr functional states through absorption of red and far-red light. The photosensory core of BphPs is composed of PAS, GAF, and PHY domains. Here, we apply FTIR spectroscopy to investigate changes in the secondary structure of Rhodopseudomonas palustris BphP2 (RpBphP2) upon Pr to Pfr photoconversion. Our results indicate conversion from a ß-sheet to an α-helical element in the so-called tongue region of the PHY domain, consistent with recent X-ray structures of Deinococcus radiodurans DrBphP in dark and light states (Takala H.; et al. Nature2014, 5, 245-248). A conserved Asp in the GAF domain that noncovalently connects with the PHY domain and a conserved Pro in the tongue region of the PHY domain are essential for the ß-sheet-to-α-helix conversion.

Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs.

Bacteriophytochromes (Bphs) are red-light photoreceptor proteins with a photosensory core that consists of three distinct domains, PAS, GAF and PHY, and covalently binds biliverdin (BV) to a conserved cysteine in the PAS domain. In a recent development, PAS-GAF variants were engineered for use as a near-infrared fluorescent marker in mammalian tissues (Tsien and co-workers, Science, 2009, 324, 804-807). Here, we report the fluorescence quantum yield and photochemistry of two highly-related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We applied ultrafast spectroscopy to wild type P3 and P2 PAS-GAF proteins and their P3 D216A, Y272F and P2 D202A PAS-GAF-PHY mutant proteins. In these mutants hydrogen-bond interactions between a conserved aspartate (Asp) which connects the BV chromophore with the PHY domains are disrupted. The excited-state lifetime of the truncated P3 and P2 PAS-GAF proteins was significantly longer than in their PAS-GAF-PHY counterparts that constitute the full photosensory core. Mutation of the conserved Asp to Ala in the PAS-GAF-PHY protein had a similar but larger effect. The fluorescence quantum yields of the P3 D216A and Y272F mutants were 0.066, higher than that of wild type P3 (0.043) and similar to the engineered Bph of Tsien and co-workers. We conclude that elimination of a key hydrogen-bond interaction between Asp and a conserved Arg in the PHY domain is responsible for the excited-state lifetime increase in all Bph variants studied here. H/D exchange resulted in a 1.4-1.7 fold increase of excited-state lifetime. The results support a reaction model in which deactivation of the BV chromophore proceeds via excited-state proton transfer from the BV pyrrole nitrogens to the backbone of the conserved Asp or to a bound water. This work may aid in rational structure- and mechanism-based conversion of constructs based on P3 and other BPhs into efficient near-IR, deep tissue, fluorescent markers.

Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy.

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C(15)═C(16) double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris , RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ∼1540 cm(-1). Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.

Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome.

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15 horizontal lineC16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.

On the signaling mechanism and the absence of photoreversibility in the AppA BLUF domain.

The flavoprotein AppA from Rhodobacter sphaeroides contains an N-terminal, FAD-binding BLUF photoreceptor domain. Upon illumination, the AppA BLUF domain forms a signaling state that is characterized by red-shifted absorbance by 10 nm, a state known as AppA(RED). We have applied ultrafast spectroscopy on the photoaccumulated AppA(RED) state to investigate the photoreversible properties of the AppA BLUF domain. On light absorption by AppA(RED), the FAD singlet excited state FAD(RED)* decays monoexponentially in 7 ps to form the neutral semiquinone radical FADH(*), which subsequently decays to the original AppA(RED) molecular ground state in 60 ps. Thus, FAD(RED)* is deactivated rapidly via electron and proton transfer, probably from the conserved tyrosine Tyr-21 to FAD, followed by radical-pair recombination. We conclude that, in contrast to many other photoreceptors, the AppA BLUF domain is not photoreversible and does not enter alternative reaction pathways upon absorption of a second photon. To explain these properties, we propose that a molecular configuration is formed upon excitation of AppA(RED) that corresponds to a forward reaction intermediate previously identified for the dark-state BLUF photoreaction. Upon excitation of AppA(RED), the BLUF domain therefore enters its forward reaction coordinate, readily re-forming the AppA(RED) ground state and suppressing reverse or side reactions. The monoexponential decay of FAD* indicates that the FAD-binding pocket in AppA(RED) is significantly more rigid than in dark-state AppA. Steady-state fluorescence experiments on wild-type, W104F, and W64F mutant BLUF domains show tryptophan fluorescence maxima that correspond with a buried conformation of Trp-104 in dark and light states. We conclude that Trp-104 does not become exposed to solvent during the BLUF photocycle.

On the role of aromatic side chains in the photoactivation of BLUF domains.

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 electron-transfer 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.