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

The photoactive yellow protein (PYP) from <i>Halorhodospira halophila</i> (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 <i>Rhodospirillum centenum</i> (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 <i>p</i>-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

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 <i>Synechocystis</i> 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 (P_r-I, P_r-II, and P_r-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 P_r-I state as the photoactive state <i>in vitro</i>, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield

Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from <i>Leptospira biflexa</i> and <i>Halorhodospira halophila</i>

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from <i>Leptospira biflexa</i> (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 <i>Halorhodospira halophila</i> (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 <i>p</i>CAH 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

Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from <i>Halorhodospira halophila</i>

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the <i>p</i>-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 <i>Halorhodospira halophila</i> 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 <i>p</i>-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results