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.

Optical Dedoping Mechanism for P3HT:F4TCNQ Mixtures.

Doping-induced solubility control (DISC) is a recently introduced photolithographic technique for semiconducting polymers, which utilizes reversible changes in polymer solubility upon doping to allow the polymer to function as its own photoresist. Central to this process is a wavelength sensitive optical dedoping reaction, which is poorly understood but generates subdiffraction-limited topographic features and provides optical control of the polymer doping level. Here, we examine the mechanism of optical dedoping in the semiconducting polymer poly-3-hexylthiophene (P3HT) doped by 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4TCNQ), via a combination of ultrafast and steady-state spectroscopy, ab initio calculations, and multidimensional NMR. A simple photoinduced back electron transfer mechanism from reduced F4TCNQ to oxidized P3HT does not explain the observed photophysics. Instead, photoexcited F4TCNQ* reacts with THF solvent molecules to form a neutral, nondoping, and highly soluble F4TCNQ-THF complex. Hence, ionized F4TCNQ is removed from the P3HT indirectly by depletion of the neutral F4TCNQ. Because the reaction involves only the dopant and similar photoreactivity would expected for most other dopant molecules, we expect optical DISC patterning should be generalizable to a wide range of polymer:dopant systems.