Energy transfer and trapping in photosystem I with and without chlorophyll-f.

We establish a general kinetic scheme for energy transfer and trapping in the photosystem I (PSI) of cyanobacteria grown under white light (WL) or far-red light (FRL) conditions. With the help of simultaneous target analysis of all emission and transient absorption datasets measured in five cyanobacterial strains, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described by Bulk Chl a, two Red Chl a, and a reaction center compartment (WL-RC). The FRL-PSI contains two additional Chl f compartments. The lowest excited state of the FRL-RC is downshifted by ≈ 29 nm. The rate of charge separation drops from ≈900 ns-1 in WL-RC to ≈300 ns-1 in FRL-RC. The delayed trapping in the FRL-PSI (≈130 ps) is explained by uphill energy transfer from the Chl f compartments with Gibbs free energies of ≈kBT below that of the FRL-RC.

Pyglotaran: a lego-like Python framework for global and target analysis of time-resolved spectra.

The dynamics of molecular systems can be studied with time-resolved spectroscopy combined with model-based analysis. A Python framework for global and target analysis of time-resolved spectra is introduced with the help of three case studies. The first study, concerning broadband absorption of intersystem crossing in 4-thiothymidine, demonstrates the framework's ability to resolve vibrational wavepackets with a time resolution of ≈10 fs using damped oscillations and their associated spectra and phases. Thereby, a parametric description of the "coherent artifact" is crucial. The second study addresses multichromophoric systems composed of two perylene bisimide chromophores. Here, pyglotaran's guidance spectra and lego-like model composition enable the integration of spectral and kinetic properties of the parent chromophores, revealing a loss process, the undesired production of a radical pair, that reduces the light harvesting efficiency. In the third, time-resolved emission case study of whole photosynthetic cells, a megacomplex containing ≈500 chromophores of five different types is described by a combination of the kinetic models for its elements. As direct fitting of the data by theoretical simulation is unfeasible, our global and target analysis methodology provides a useful 'middle ground' where the theoretical description and the fit of the experimental data can meet. The pyglotaran framework enables the lego-like creation of kinetic models through its modular design and seamless integration with the rich Python ecosystem, particularly Jupyter notebooks. With extensive documentation and a robust validation framework, pyglotaran ensures accessibility and reliability for researchers, serving as an invaluable tool for understanding complex molecular systems.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive 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.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Confinement in crystal lattice alters entire photocycle pathway of the Photoactive Yellow Protein.

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.

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.

Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy.

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a ß-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 µs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 µs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the ß-sheet, and an additional helical element. The second phase occurs in approximately 240 µs. The final spectrum at 500 µs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.

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.