Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

The Orange Carotenoid Protein Triggers Cyanobacterial Photoprotection by Quenching Bilins via a Structural Switch of Its Carotenoid.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

QM calculations predict the energetics and infrared spectra of transient glutamine isomers in LOV photoreceptors.

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

Vibronic dynamics resolved by global and target analysis of ultrafast transient absorption spectra.

We present a methodology that provides a complete parametric description of the time evolution of the electronically and vibrationally excited states as detected by ultrafast transient absorption (TA). Differently from previous approaches, which started fitting the data after ≈100 fs, no data are left out in our methodology, and the "coherent artifact" and the instrument response function are fully taken into account. In case studies, the method is applied to solvents, the dye Nile blue, and all-trans ß-carotene in cyclohexane solution. The estimated Damped Oscillation Associated Spectra (DOAS) and phases express the most important vibrational frequencies present in the molecular system. By global fit alone of the experimental data, it is difficult to interpret in detail the underlying dynamics. Since it is unfeasible to directly fit the data by a theoretical simulation, our enhanced DOAS methodology thus provides a useful "middle ground" where the theoretical description and the fit of the experimental data can meet. ß-carotene in cyclohexane was complementarily studied with femtosecond stimulated Raman spectroscopy (FSRS). The fs-ps dynamics of ß-carotene in cyclohexane in TA and FSRS experiments can be described by a sequential scheme S2 → hot S1 → S1' → S1 → S0 with lifetimes of 167 fs (fixed), 0.35, 1.1, and 9.6 ps. The correspondence of DOAS decaying concomitantly with hot S1 and the Species Associated Difference Spectra of hot S1 in TA and FSRS suggest that we observe here features of the vibrational relaxation and nuclear reorganization responsible for the hot S1 to S1 transition.

Dual Photoisomerization on Distinct Potential Energy Surfaces in a UV-Absorbing Rhodopsin.

UV-absorbing rhodopsins are essential for UV vision and sensing in all kingdoms of life. Unlike the well-known visible-absorbing rhodopsins, which bind a protonated retinal Schiff base for light absorption, UV-absorbing rhodopsins bind an unprotonated retinal Schiff base. Thus far, the photoreaction dynamics and mechanisms of UV-absorbing rhodopsins have remained essentially unknown. Here, we report the complete excited- and ground-state dynamics of the UV form of histidine kinase rhodopsin 1 (HKR1) from eukaryotic algae, using femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy, covering time scales from femtoseconds to milliseconds. We found that energy-level ordering is inverted with respect to visible-absorbing rhodopsins, with an optically forbidden low-lying S1 excited state that has Ag- symmetry and a higher-lying UV-absorbing S2 state of Bu+ symmetry. UV-photoexcitation to the S2 state elicits a unique dual-isomerization reaction: first, C13═C14 cis-trans isomerization occurs during S2-S1 evolution in <100 fs. This very fast reaction features the remarkable property that the newly formed isomer appears in the excited state rather than in the ground state. Second, C15═N16 anti-syn isomerization occurs on the S1-S0 evolution to the ground state in 4.8 ps. We detected two ground-state unprotonated retinal photoproducts, 13-trans/15-anti (all-trans) and 13-cis/15-syn, after relaxation to the ground state. These isomers become protonated in 58 µs and 3.2 ms, respectively, resulting in formation of the blue-absorbing form of HKR1. Our results constitute a benchmark of UV-induced photochemistry of animal and microbial rhodopsins.

Unraveling the Excited-State Dynamics and Light-Harvesting Functions of Xanthophylls in Light-Harvesting Complex II Using Femtosecond Stimulated Raman Spectroscopy.

Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.

Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

The terminal phycobilisome emitter, LCM: A light-harvesting pigment with a phytochrome chromophore.

Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L(CM). The chromophore domain of L(CM) forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L(CM) by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L(CM). Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

The femtosecond-to-second photochemistry of red-shifted fast-closing anion channelrhodopsin PsACR1.

Anion channelrhodopsins (ACRs) are of great interest due to their ability to inhibit electrical signaling in optogenetic experiments. The photochemistry of ACRs is currently poorly understood and an improved understanding would be beneficial for rational design of ACRs with modified properties. Activation/deactivation of ACRs involves a series of photoreactions ranging from femtoseconds to seconds, thus real-time observation is essential to comprehend the full complexity of the photochemical processes. Here we investigate the photocycle of an ACR from Proteomonas sulcata (PsACR1), which is valuable for optogenetic applications due to the red-shifted absorption and action spectra compared to the prototype ACRs from Guillardia theta: GtACR1 and GtACR2, and the fast channel closing properties. From femto-to-submillisecond transient absorption spectroscopy, flash photolysis, and point mutations of acidic residues near the retinal Schiff base (RSB), E64, and D230, we found that the photoisomerization occurs in ∼500 fs independent of the protonation state of E64. Notably, E64 is involved in the rearrangement of the hydrogen-bond network near the RSB after photoisomerization. Furthermore, we suggest that E64 works as a primary proton acceptor during deprotonation of the RSB as has been proposed for GtACR1. Our findings allow for a deeper understanding of the photochemistry on the activation/deactivation of ACRs.

Spectral watermarking in femtosecond stimulated Raman spectroscopy: resolving the nature of the carotenoid S* state.

A new method for recording femtosecond stimulated Raman spectra was developed that dramatically improves and automatizes baseline problems. Instead of using a narrowband Raman source, the experiment is performed using shaping of a broadband source. This allows locking the signal into carefully crafted watermarks that can be recovered from measured data with high fidelity. The approach uses unique properties of Raman scattering, thus allowing a direct recording of stimulated Raman signals with robust rejection of baselines and fixed-pattern-noise. Low cost technology for generating required pulse-shapes was developed and demonstrated. The methodology is applicable to any Raman experiment but primarily targets Femtosecond Stimulated Raman spectroscopy (FSRS) where a lack of robust methods for parasitic signal rejection has been a major obstacle in the practical development of the field in the last decade. The delivered improvement in FSRS experiments was demonstrated by recording evidence that the so-called S* state of carotenoids in solution corresponds to the optically forbidden S1 state of a sparsely populated carotenoid conformation.

The photochemistry of sodium ion pump rhodopsin observed by watermarked femto- to submillisecond stimulated Raman spectroscopy.

Krokinobacter rhodopsin 2 (KR2) is a recently discovered light-driven Na(+) pump that holds significant promise for application as a neural silencer in optogenetics. KR2 transports Na(+) (in NaCl solution) or H(+) (in larger cation solution, e.g. in CsCl) during its photocycle. Here, we investigate the photochemistry of KR2 with the recently developed watermarked, baseline-free femto- to submillisecond transient stimulated Raman spectroscopy (TSRS), which enables us to investigate retinal chromophore dynamics in real time with high spectral resolution over a large time range. We propose a new photocycle from femtoseconds to submilliseconds: J (formed in ∼200 fs) → K (∼3 ps) → K/L1 (∼20 ps) → K/L2 (∼30 ns) → L/M (∼20 µs). KR2 binds a Na(+) ion that is not transported on the extracellular side, of which the function is unclear. We demonstrate with TSRS that for the D102N mutant in NaCl (with Na(+) unbound, Na(+) transport) and for WT KR2 in CsCl (with Na(+) unbound, H(+) transport), the extracellular Na(+) binding significantly influences the intermediate K/L/M state equilibrium on the photocycle, while the identity of the transported ion, Na(+) or H(+), does not affect the photocycle. Our findings will contribute to further elucidation of the molecular mechanisms of KR2.

Proton-Coupled Electron Transfer Constitutes the Photoactivation Mechanism of the Plant Photoreceptor UVR8.

UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

Photoinduced transformation of UVR8 monitored by vibrational and fluorescence spectroscopy.

Tryptophan residues at the dimer interface of the plant photoreceptor UVR8 promote monomerisation after UV-B absorption via a so far unknown mechanism. Using FTIR spectroscopy we assign light-induced structural transitions of UVR8 mainly to amino acid side chains without major transformations of the secondary structure of the physiologically relevant C-terminal extension. Additionally, we assign the monomerisation associated increase and red shift of the UVR8 tryptophan emission to a photoinduced rearrangement of tryptophan side chains and a relocation of the aspartic acid residues D96 and D107, respectively. By illumination dependent emission spectroscopy we furthermore determined the quantum yield of photoinduced monomerisation to 20 ± 8%.

Kinetic isotope effect of proton-coupled electron transfer in a hydrogen bonded phenol-pyrrolidino[60]fullerene.

Proton-coupled electron transfer (PCET) plays a central role in photosynthesis and potentially in solar-to-fuel systems. We report a spectroscopy study on a phenol-pyrrolidino[60]fullerene. Quenching of the singlet excited state from 1 ns to 250 ps is assigned to PCET. A H/D exchange study reveals a kinetic isotope effect (KIE) of 3.0, consistent with a concerted PCET mechanism.

Triplet-triplet annihilation upconversion followed by FRET for the red light activation of a photodissociative ruthenium complex in liposomes.

Upconversion is a promising way to trigger high-energy photochemistry with low-energy photons. However, combining upconversion schemes with non-radiative energy transfer is challenging because bringing several photochemically active components in close proximity results in complex multi-component systems where quenching processes may deactivate the whole assembly. In this work, PEGylated liposomes were prepared that contained three photoactive components: a porphyrin dye absorbing red light, a perylene moiety emitting in the blue, and a light-activatable ruthenium prodrug sensitive to blue light. Time-dependent spectroscopic studies demonstrate that singlet perylene excited states are non-radiatively transferred to the nearby ruthenium complex by Förster resonance energy transfer (FRET). Under red-light irradiation of the three-component membranes, triplet-triplet annihilation upconversion (TTA-UC) occurs followed by FRET, which results in a more efficient activation of the ruthenium prodrug compared to a physical mixture of two-component upconverting liposomes and liposomes containing only the ruthenium complex. This work represents a rare example where TTA-UC and Förster resonance energy transfer are combined to achieve prodrug activation in the phototherapeutic window.

Key residues for the light regulation of the blue light-activated adenylyl cyclase from Beggiatoa sp.

Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand ß5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand ß5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.

Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane pyrimidine dimer photolyase α-helices.

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure-function study by FTIR spectroscopy of the Escherichia coli cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.

Synthesis and photophysics of a red-light absorbing supramolecular chromophore system.

In search of supramolecular antenna systems for light-harvesting applications, we report on a short and effective synthesis of a fused NDI-zinc-salphen-based chromophore (salphen = bis-salicylimide phenylene) and its photophysical properties. A supramolecular recognition motif is embedded into the chromophoric π-system of this compound. The fused π-chromophore behaves as one pigment, absorbs light between 600 and 750 nm and displays a modest Stokes shift. Upon binding pyridines, the compound (DATZnS) does not change its redox potentials, does not undergo any internal excited state quenching and does not appreciably alter its excited state lifetime. These notable properties define DATZnS as an alternative to porphyrin-based components used in supramolecular light-harvesting architectures.

Two-photon Absorption and Photoionization of a Bacterial Phytochrome.

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of µJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.