Not All Photoactive Yellow Proteins Are Built Alike: Surprises and Insights into Chromophore Photoisomerization, Protonation, and Thermal Reisomerization of the Photoactive Yellow Protein Isolated from Salinibacter ruber.

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.

Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira halophila.

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.

Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira halophila.

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.

Hot Carrier Cooling and Recombination Dynamics of Chlorine-Doped Hybrid Perovskite Single Crystals.

Controlling the photoexcited properties and behavior of hybrid perovskites by halide doping has the potential to impact a wide range of emerging technologies, including solar cells and radiation detectors. Crystalline samples of methylammonium lead bromide substituted with chlorine (MAPbBr3-xClx) were examined by transient reflectivity spectroscopy and nonadiabatic molecular dynamics simulations. At picosecond time scales, the addition of chlorine to the perovskite crystal increased the observed rate of hot carrier cooling and the calculated electron-phonon coupling constants. Chlorine-doped samples also exhibit a slower surface recombination velocity and a smaller ambipolar mobility.

Ultrafast Spintronics: Dynamics of the Photoisomerization-Induced Spin-Charge Excited-State (PISCES) Mechanism in Spirooxazine-Based Photomagnetic Materials.

The optical control of spin state is of interest in the development of spintronic materials for data processing and storage technologies. Photomagnetic effects at the single-molecule level have recently been observed in the thin film state at 300 K in photochromic cobalt dioxolenes. Visible light excitation leads to ring-closure of a photochromic spirooxazine bound to a cobalt dioxolene, which leads to generation of a high magnetization state. Formation of the photomagnetic state occurs through a photoisomerization-induced spin-charge excited-state process and is dictated by the spirooxazine ligand dynamics. Here, we report a mechanistic investigation by ultrafast spectroscopy in the UV-vis region of the photochemical ring-closing process in the parent spirooxazine, azahomoadamantylphenanthroline spirooxazine, and the photomagnetic spirooxazine cobalt-dioxolene complex. The cobalt appears to stabilize a photomerocycanine transient intermediate, presumably the TCC isomer, formed along the ground-state potential energy surface (PES). Structural changes associated with the TCC isomer induces formation of the high-spin Co(II) form, suggesting that magnetization dymanics can occur along the excited-state PES, leading to ultrafast switching on the ps time scale. We demonstrate the full ring closure of the spiro-oxazine ligand is not required to switch magnetization states which can be induced with a higher yielding isomerization reaction. The ability of this system to undergo optically induced spin state switching on the ps time scale in the solid state makes it a promising canididate for resistive nonvolatile memory technologies.

Protonation Heterogeneity Modulates the Ultrafast Photocycle Initiation Dynamics of Phytochrome Cph1.

Phytochrome proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from Synechocystis sp. PCC6803 (Cph1Δ) were resolved with a dual-excitation-wavelength-interleaved pump-probe (DEWI) approach with two excitation wavelengths (600 and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (Pr-I, Pr-II, and Pr-III), each composed of multiple kinetically distinct subpopulations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength. Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low-temperature Pr-I state as the photoactive state in vitro, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield.

Noncanonical Photocycle Initiation Dynamics of the Photoactive Yellow Protein (PYP) Domain of the PYP-Phytochrome-Related (Ppr) Photoreceptor.

The photoactive yellow protein (PYP) from Halorhodospira halophila (Hhal) is a bacterial photoreceptor and model system for exploring functional protein dynamics. We report ultrafast spectroscopy experiments that probe photocycle initiation dynamics in the PYP domain from the multidomain PYP-phytochrome-related photoreceptor from Rhodospirillum centenum (Rcen). As with Hhal PYP, Rcen PYP exhibits similar excited-state dynamics; in contrast, Rcen PYP exhibits altered photoproduct ground-state dynamics in which the primary I0 intermediate as observed in Hhal PYP is absent. This property is attributed to a tighter, more sterically constrained binding pocket around the p-coumaric acid chromophore due to a change in the Rcen PYP protein structure that places Phe98 instead of Met100 in contact with the chromophore. Hence, the I0 state is not a necessary step for the initiation of productive PYP photocycles and the ubiquitously studied Hhal PYP may not be representative of the broader PYP family of photodynamics.