Ultrafast proton coupled electron transfer (PCET) dynamics in 9-anthranol-aliphatic amine system

Femtosecond infrared absorption studies strongly suggest that photoexcited 9-anthranol takes part in an ultrafast electron transfer (ET) reaction in electron-donating triethylamine solvent, but that ultrafast proton coupled electron transfer (PCET) occurs in diethylamine solvent

Direct Femtosecond Observation of Tight and Loose Ion Pairs upon Photoinduced Bimolecular Electron Transfer

Polarisationsempfindliche ultraschnelle Infrarotmessungen des photoinduzierten Elektronentransfers in Donor-Akzeptor-Paaren in polarem Acetonitril können individuelle Beiträge locker und fest gebundener Ionenpaare detektieren. Die hoch anisotropen Signale der zweiten verdeutlichen, dass die gegenseitige Orientierung der Reaktionspartner wichtig ist (siehe Bild), d. h., bisherige theoretische Modelle, die nur die Abstände zwischen sphärischen Spezies berücksichtigen, müssen verfeinert werden

Experimental observation of nanophase segregation of aqueous salt solutions around the predicted liquid-liquid transition in water

The liquid-liquid transition in supercooled liquid water, predicted to occur around 220 K, is controversial due to the difficulty of studying it caused by competition from ice crystallization (the so-called “no man’s land”). In aqueous solutions, it has been predicted to give rise to phase separation on a nanometer scale between a solute-rich high-density phase and a water-rich low-density phase. Here we report direct experimental evidence for the formation of a nanosegregated phase in eutectic aqueous solutions of LiCl and LiSCN where the presence of crystalline water can be experimentally excluded. Femtosecond infrared and Raman spectroscopies are used to determine the temperature-dependent structuring of water, the solvation of the SCN- anion, and the size of the phase segregated domains

Ultrafast Light-Driven Electron Transfer in a Ru(II)tris(bipyridine)-Labelled Multiheme Cytochrome

Multiheme cytochromes attract much attention for their electron transport properties. These proteins conduct electrons across bacterial cell walls, along extracellular filaments, and when purified can serve as bionanoelectronic junctions. Thus, it is important and necessary to identify and understand the factors governing electron transfer in this family of proteins. To this end we have used ultra-fast transient absorbance spectroscopy, to define heme-heme electron transfer dynamics in the representative multiheme cytochrome STC from Shewanella oneidensis in aqueous solution. STC was photo-sensitized by site-selective labelling with a Ru(II)(bipyridine)3 dye and the dynamics of light-driven electron transfer described by a kinetic model corroborated by molecular dynamics simulation and density functional theory calculations. With the dye attached adjacent to STC Heme IV, a rate constant of 87 x 106 s-1 was resolved for Heme IV → Heme III electron transfer. With the dye attached adjacent to STC Heme I, at the opposite terminus of the tetraheme chain, a rate constant of 125 x 106 s-1 was defined for Heme I → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90o with edge-edge (Fe-Fe) distances of ≈6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve inter-heme electron transfer in Ru-labeled multiheme cytochromes

Site Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

Real-time observation of structure change associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, PixD and AppABLUF; both proteins retain the red shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared (TRIR) difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins, and a differential response of the two sites to chromophore excitation

Unraveling the photoactivation mechanism of a light activated adenylyl cyclase using ultrafast spectroscopy coupled with unnatural amino acid mutagenesis

The hydrogen bonding network that surrounds the flavin in Blue Light Utilizing FAD (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. The network contains a highly conserved tyrosine that is essential for photoactivation. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid (UAA) incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase (AC) from Oscillatoria Acuminata (OaPAC). Our work elucidates the direct connection between the photoactivation process in the BLUF domain and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate formation of FADH● as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogs to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FAD● ̶. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the AC domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP to cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FAD● ̶. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in adenylyl cyclase activity

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor:A Glutamine Lever Induces Unfolding of the Jα Helix

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 μs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone C═O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function