Ultrafast Charge-Transfer Dynamics in the Iron–Sulfur Complex of <i>Rhodobacter capsulatus</i> Ferredoxin VI

Iron–sulfur proteins play essential roles in various biological processes. Their electronic structure and vibrational dynamics are key to their rich chemistry but nontrivial to unravel. Here, the first ultrafast transient absorption and impulsive coherent vibrational spectroscopic (ICVS) studies on 2Fe–2S clusters in <i>Rhodobacter capsulatus</i> ferreodoxin VI are characterized. Photoexcitation initiated populations on multiple excited electronic states that evolve into each other in a long-lived charge-transfer state. This suggests a potential light-induced electron-transfer pathway as well as the possibility of using iron–sulfur proteins as photosensitizers for light-dependent enzymes. A tyrosine chain near the active site suggests potential hole-transfer pathways and affirms this electron-transfer pathway. The ICVS data revealed vibrational bands at 417 and 484 cm^–1, with the latter attributed to an excited-state mode. The temperature dependence of the ICVS modes suggests that the temperature effect on protein structure or conformational heterogeneities needs to be considered during cryogenic temperature studies

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

Excited-State Self-Trapping and Ground-State Relaxation Dynamics in Poly(3-hexylthiophene) Resolved with Broadband Pump–Dump–Probe Spectroscopy

Broadband femtosecond transient absorption spectroscopy is used to explore the mechanisms underlying excited-state and ground-state exciton relaxation in poly(3-hexylthiophene) (P3HT) solution. We focus on the picosecond spectral shifts in the ground and excited states of P3HT, using pump–probe (PP) and pump–dump–probe (PDP) techniques to investigate exciton relaxation mechanisms. Excited-state PP signals resolved a dynamic stimulated emission Stokes shift and ground-state reorganization; PDP signals resolved a blue-shifting nonequilibrium ground-state bleach. Initial structural reorganization is shown to be faster in the excited state. Ground-state reorganization is shown to be dependent on dump time, with later times resulting in relatively more population undergoing slow (∼20 ps) reorganization. These observations are discussed in the context of structural relaxation involving small-scale (<1 ps) and large-scale (>1 ps) planarization of thiophene groups following photoexcitation. Excited-state and ground-state dynamics are contrasted in terms of electronic structure defining the torsional potential energy surfaces. It is shown that the primary excitonic relaxation mechanism is excited-state self-trapping via torsional relaxation rather than exciton energy transfer

Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from <i>Nostoc punctiforme</i>. 2. Reverse Dynamics

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P_r and P_fr, respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15<i>Z</i> ground state, like the familiar P_r state of phytochromes, and a green-absorbing photoproduct (^15<i>E</i>P_g). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium <i>Nostoc punctiforme</i> to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily

Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from <i>Nostoc punctiforme</i>. 1. Forward Dynamics

Phytochromes are well-known red/far-red photosensory proteins that utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to detect the ratio of red to far-red light. Cyanobacteriochromes (CBCRs) are related photosensory proteins with a bilin-binding GAF domain, but much more diverse spectral sensitivity, with five recognized subfamilies of CBCRs described to date. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing ground state, like the familiar P_r state of phytochromes, and a green-absorbing photoproduct (P_g). Here, we examine the ultrafast forward photodynamics of the red/green CBCR NpR6012g4 from the <i>NpR6012</i> locus of the nitrogen-fixing cyanobacterium <i>Nostoc punctiforme</i>. Using transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics that induces the forward reaction (red-absorbing to green-absorbing), which we interpret as arising from ground-state heterogeneity. Excited-state decays with lifetimes of 55 and 345 ps generate the primary photoproduct (Lumi-R), and the fastest decay (5 ps) did not produce Lumi-R. Although the photoinduced kinetics of Npr6012g4 is comparable with that of the Cph1 phytochrome isolated from <i>Synechocystis</i> cyanobacteria, NpR6012g4 exhibits a ≥2–3-fold higher photochemical quantum yield. Understanding the structural basis of this enhanced quantum yield may prove to be useful in increasing the photochemical efficiency of other bilin-based photosensors

Optically Guided Photoactivity: Coordinating Tautomerization, Photoisomerization, Inhomogeneity, and Reactive Intermediates within the RcaE Cyanobacteriochrome

The RcaE cyanobacteriochrome uses a linear tetrapyrrole chromophore to sense the ratio of green and red light to enable the <i>Fremyella diplosiphon</i> cyanobacterium to control the expression of the photosynthetic infrastructure for efficient utilization of incident light. The femtosecond photodynamics of the embedded phycocyanobilin chromophore within RcaE were characterized with dispersed femtosecond pump–dump–probe spectroscopy, which resolved a complex interplay of excited-state proton transfer, photoisomerization, multilayered inhomogeneity, and reactive intermediates. These reactions were integrated within a central model that incorporated a rapid (200 fs) excited-state Le Châtelier redistribution between parallel evolving populations ascribed to different tautomers. Three photoproducts were resolved and originates from four independent subpopulations, each with different dump-induced behavior: Lumi-G_o was depleted, Lumi-G_r was unaffected, and Lumi-G_f was enhanced. This suggests that RcaE may be engineered to act either as an <i>in vivo</i> fluorescent probe (after single-pump excitation) or as an <i>in vivo</i> optogenetic sample (after pump and dump excitation)

Reactive Ground-State Pathways Are Not Ubiquitous in Red/Green Cyanobacteriochromes

Recent characterization of the red/green cyanobacteriochrome (CBCR) NpR6012g4 revealed a high quantum yield for its forward photoreaction [J. Am. Chem. Soc. 2012, 134, 130−133] that was ascribed to the activity of hidden, productive ground-state intermediates. The dynamics of the pathways involving these ground-state intermediates was resolved with femtosecond dispersed pump–dump–probe spectroscopy, the first such study reported for any CBCR. To address the ubiquity of such second-chance initiation dynamics (SCID) in CBCRs, we examined the closely related red/green CBCR NpF2164g6 from <i>Nostoc punctiforme</i>. Both NpF2164g6 and NpR6012g4 use phycocyanobilin as the chromophore precursor and exhibit similar excited-state dynamics. However, NpF2164g6 exhibits a lower quantum yield of 32% for the generation of the isomerized Lumi-R primary photoproduct, compared to 40% for NpR6012g4. This difference arises from significantly different ground-state dynamics between the two proteins, with the SCID mechanism deactivated in NpF2164g6. We present an integrated inhomogeneous target model that self-consistently fits the pump–probe and pump–dump–probe signals for both forward and reverse photoreactions in both proteins. This work demonstrates that reactive ground-state intermediates are not ubiquitous phenomena in CBCRs

Sequestering High-Energy Electrons to Facilitate Photocatalytic Hydrogen Generation in CdSe/CdS Nanocrystals

The photocatalytic H₂O splitting activities of CdSe and CdSe/CdS core/shell quantum dots are contrasted. CdSe/CdS core/shell quantum dots constructed from 4.0 nm CdSe quantum dots are shown to be strongly active for visible-light-driven photocatalytic H₂ evolution in 0.1 M Na₂S/Na₂SO₃ solution with a turnover number of 9.94 after 5 h at 103.9 μmol/h. CdSe quantum dots themselves are only marginally active in 0.1 M Na₂S/Na₂SO₃ solution with a turnover number of 1.10 after 5 h at 11.53 μmol/h, while CdSe quantum dots in pure H₂O are found to be completely inactive. Broad-band transient absorption spectroscopy is used to elucidate the mechanisms that facilitate the enhancement in the CdSe core/shell quantum dots, which is attributed to passivation of surface-deep trap states with energies lying below the reduction potential necessary for H₂O reduction. Thus, surface trapping dynamics and energetics can be manipulated to dictate the photocatalytic activities of novel CdSe quantum dot based photocatalytic materials

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