Frequency-Domain Spectroscopic Study of the Photosystem I Supercomplexes, Isolated IsiA Monomers, and the Intact IsiA Ring.

The PSI3-IsiA18 supercomplex is one of the largest and most complicated assemblies in photosynthesis. The IsiA ring, composed of 18 IsiA monomers (IsiA18) surrounding the PSI trimer (PSI3), forms under iron-deficient conditions in cyanobacteria and acts as a peripheral antenna. Based on the supercomplex structure recently determined via cryo-EM imaging, we model various optical spectra of the IsiA monomers and IsiA18 ring. Comparison of the absorption and emission spectra of the isolated IsiA monomers and the full ring reveals that about 2.7 chlorophylls (Chls) are lost in the isolated IsiA monomers. The best fits for isolated monomers spectra are obtained assuming the absence of Chl 508 and Chl 517 and 70% loss of Chl 511. The best model describing all three hexamers and the entire ring suggests that the lowest energy pigments are Chls 511, 514, and 517. Based on the modeling results presented in this work, we conclude that there are most likely three entry points for EET from the IsiA6 hexamer to the PSI core monomer, with two of these entry points likely being located next to each other (i.e., nine entry points from IsiA18 to the PSI3 trimer). Finally, we show that excitation energy transfer inside individual monomers is fast (<2 ps at T = 5 K) and at least 20 times faster than intermonomer energy transfer.

Exciton Lifetime Distributions and Population Dynamics in the FMO Protein Complex from Prosthecochloris aestuarii.

Significant protein rearrangement upon excitation and energy transfer in Fenna-Matthews-Olson protein of Prosthecochloris aestuarii results in a modified energy landscape, which induces more changes in pigment site energies than predicted by the "standard" hole-burning theory. The energy changes are elucidated by simulations while investigating the effects of site-dependent disorder, both static (site-energy distribution widths) and dynamic (spectral density shapes). The resulting optimized site energies and their fluctuations are consistent with relative differences observed in inhomogeneous widths calculated by recent molecular dynamic simulations. Two sets of different spectral densities reveal how their shapes affect the population dynamics and distribution of exciton lifetimes. Calculations revealed the wavelength-dependent distributions of exciton lifetimes (T 1) in the femtosecond to picosecond time frame. We suggest that the calculated multimodal and asymmetric wavelength-dependent T 1 distributions offer more insight into the interpretation of resonant hole-burned (HB) spectra, kinetic traces in two-dimensional (2D) electronic spectroscopy experiments, and widely used global analyses in fitting data from transient absorption experiments.

Author Correction: The structure of a red-shifted photosystem I reveals a red site in the core antenna.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19953-w.

The structure of a red-shifted photosystem I reveals a red site in the core antenna.

Photosystem I coordinates more than 90 chlorophylls in its core antenna while achieving near perfect quantum efficiency. Low energy chlorophylls (also known as red chlorophylls) residing in the antenna are important for energy transfer dynamics and yield, however, their precise location remained elusive. Here, we construct a chimeric Photosystem I complex in Synechocystis PCC 6803 that shows enhanced absorption in the red spectral region. We combine Cryo-EM and spectroscopy to determine the structure-function relationship in this red-shifted Photosystem I complex. Determining the structure of this complex reveals the precise architecture of the low energy site as well as large scale structural heterogeneity which is probably universal to all trimeric Photosystem I complexes. Identifying the structural elements that constitute red sites can expand the absorption spectrum of oxygenic photosynthetic and potentially modulate light harvesting efficiency.

On the Red Antenna States of Photosystem I Mutants from Cyanobacteria Synechocystis PCC 6803.

To identify the molecular composition of the low-energy states in cyanobacterial Photosystem I (PSI) of Synechocystis PCC6803, we focus on high-resolution (low-temperature) absorption, emission, resonant, and nonresonant hole-burned spectra obtained for wild-type (WT) PSI and three PSI mutants. In the Red_a mutant, the B33 chlorophyll (Chl) is added to the B31-B32 dimer; in Red_b, histidine 95 (His95) on PsaB (which coordinates Mg in the B7 Chl within the His95-B7-A31-A32-cluster) is replaced with glutamine (Gln), while in the Red_ab mutant, both mutations are made. We show that the C706 state (B31-B32) changes to the C710 state (B31-B32-B33) in both Red_a and Red_ab mutants, while the C707 state in WT Synechocystis (localized on the His95-B7-A31-A32 cluster) is modified to C716 in both Red_b and Red_ab. Excitation energy transfer from C706 to the C714 trap in the WT PSI and Red_b mutant is hampered as reflected by a weak emission at 712 nm. Large electron-phonon coupling strength (exposed via resonant hole-burned spectra) is consistent with a strong mixing of excited states with intermolecular charge transfer states leading to significantly red-shifted emission spectra. We conclude that excitation energy transfer in PSI is controlled by fine-tuning the electronic states of a small number of highly conserved red states. Finally, we show that mutations modify the protein potential energy landscape as revealed by different shapes and shifts of the blue- and red-shifted antiholes.

Influence of Hydrogen Bonds on the Electron-Phonon Coupling Strength/Marker Mode Structure and Charge Separation Rates in Reaction Centers from Rhodobacter sphaeroides.

Low-temperature persistent and transient hole-burning (HB) spectra are presented for the triple hydrogen-bonded L131LH + M160LH + M197FH mutant of Rhodobacter sphaeroides. These spectra expose the heterogeneous nature of the P-, B-, and H-bands, consistent with a distribution of electron transfer (ET) times and excitation energy transfer (EET) rates. Transient P+QA- holes are observed for fast (tens of picoseconds or faster) ET times and reveal strong coupling to phonons and marker mode(s), while the persistent holes are bleached in a fraction of reaction centers with long-lived excited states characterized by much weaker electron-phonon coupling. Exposed differences in electron-phonon coupling strength, as well as a different coupling to the marker mode(s), appear to affect the ET times. Both resonantly and nonresonantly burned persistent HB spectra show weak blue- (∼150 cm-1) and large, red-shifted (∼300 cm-1) antiholes of the P band. Slower EET times from the H- and B-bands to the special pair dimer provide new insight on the influence of hydrogen bonds on mutation-induced heterogeneity.

On uncorrelated inter-monomer Förster energy transfer in Fenna-Matthews-Olson complexes.

The Fenna-Matthews-Olson (FMO) light-harvesting antenna protein of green sulfur bacteria is a long-studied pigment-protein complex which funnels energy from the chlorosome to the reaction centre where photochemistry takes place. The structure of the FMO protein from Chlorobaculum tepidum is known as a homotrimeric complex containing eight bacteriochlorophyll a per monomer. Owing to this structure FMO has strong intra-monomer and weak inter-monomer electronic coupling constants. While long-lived (sub-picosecond) coherences within a monomer have been a prevalent topic of study over the past decade, various experimental evidence supports the presence of subsequent inter-monomer energy transfer on a picosecond time scale. The latter has been neglected by most authors in recent years by considering only sub-picosecond time scales or assuming that the inter-monomer coupling between low-energy states is too weak to warrant consideration of the entire trimer. However, Förster theory predicts that energy transfer of the order of picoseconds is possible even for very weak (less than 5 cm-1) electronic coupling between chromophores. This work reviews experimental data (with a focus on emission and hole-burned spectra) and simulations of exciton dynamics which demonstrate inter-monomer energy transfer. It is shown that the lowest energy 825 nm absorbance band cannot be properly described by a single excitonic state. The energy transfer through FMO is modelled by generalized Förster theory using a non-Markovian, reduced density matrix approach to describe the electronic structure. The disorder-averaged inter-monomer transfer time across the 825 nm band is about 27 ps. While only isolated FMO proteins are presented, the presence of inter-monomer energy transfer in the context of the overall photosystem is also briefly discussed.

Mixed Upper Exciton State of the Special Pair in Bacterial Reaction Centers.

Excitonic interactions between two closely separated bacteriochlorophyll a molecules (BChls) in the special pair of the reaction center (RC) of purple bacteria determine the positions and relative oscillator strengths of its two excitonic components. While the absorption of the lower excitonic band is well-defined, the position and the intensity of the upper excitonic band ( PY+) are still under debate. Recent 77 K two-dimensional electronic spectroscopy data on Rba. capsulatus suggested that the PY+ component absorbs at ∼840 nm, i.e., at a significantly lower energy than previously suggested. In the present work, we argue that the PY+ state is mixed with the excited states of the accessory BChls ( B*/ P Y+) leading to excitons contributing to the 785-825 nm spectral region which is consistent with previously published data. This conclusion is based on hole-burning/linear dichroism data and modeling studies of the excitonic structure of the RC using a non-Markovian reduced density matrix approach.

Dichotomous Disorder versus Excitonic Splitting of the B800 Band of Allochromatium vinosum.

The LH2 antenna complex of the purple bacterium Allochromatium vinosum has a distinct double peak structure of the 800 nm band (B800). Several hypotheses were proposed to explain its origin. Recent 77 K two-dimensional electronic spectroscopy data suggested that excitonic coupling of dimerized bacteriochlorophylls (BChls) within the B800 ring is largely responsible for the B800 split [M. Schröter et al., J. Phys. Chem. Lett. 2018, 9, 1340]. Here we argue that the excitonic interactions between BChls in the B800 ring, though present, are weak and cannot explain the B800 band split. This conclusion is based on hole-burning data and modeling studies using an exciton model with dichotomous protein conformation disorder. Therefore, we uphold our earlier interpretation, first reported by Kell et al. [ J. Phys. Chem. B 2017, 121, 9999], that the two B800 sub-bands are due to different site-energies (most likely due to weakly and strongly hydrogen-bonded B800 BChls).

Impact of Single-Point Mutations on the Excitonic Structure and Dynamics in a Fenna-Matthews-Olson Complex.

Hole burning (HB) spectroscopy and modeling studies reveal significant changes in the excitonic structure and dynamics in several mutants of the FMO trimer from the Chlorobaculum tepidum. The excited-state decay times ( T1) of the high-energy excitons are significantly modified when mutation occurs near bacteriochlorophyll (BChl) 1 (V152N mutant) or BChl 6 (W184F). Longer (averaged) T1 times of highest-energy excitons in V152N and W184F mutants suggest that site energies of BChls 1 and 6, believed to play an important role in receiving excitation from the baseplate BChls, likely play a critical role to ensure the femtosecond (fs) energy relaxation observed in wild-type FMO. HB spectroscopy reveals preferentially slower T1 times (about 1 ps on average) because fs times prohibit HB due to an extremely low HB quantum yield. Uncorrelated (incoherent) excitation energy transfer times between monomers, the composition of exciton states, and average, frequency-dependent, excited-state decay times ( T1) are discussed.

Structure-Based Exciton Hamiltonian and Dynamics for the Reconstituted Wild-type CP29 Protein Antenna Complex of the Photosystem II.

We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls a and 3 ± 0.6 Chls b per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls a and 3.5 Chls b revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl b614 is lost during the reconstitution/purification procedure, whereas Chls a are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl a604) is easily burned with λB = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the a611, a612, a615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl a604 site energy observed in the minor sub. II occurs only in reconstituted complexes.

Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum Fenna-Matthews-Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies.

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub821 and Sub817) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

Energy landscape of the intact and destabilized FMO antennas from C. tepidum and the L122Q mutant: Low temperature spectroscopy and modeling study.

We discuss the excitonic energy landscape of the typically studied wild-type (WT) Fenna-Matthews-Olson (FMO) antenna protein from the green sulfur bacterium Chlorobaculum tepidum (referred to as WTM), which is described as a mixture of intact (WTI) and destabilized (WTD) complexes. Optical spectra of WTM and the L122Q mutant (where leucine 122 near BChl 8 is replaced with glutamine) are compared to WTI FMO. We show that WTM and L122Q samples are mixtures of two subpopulations of proteins, most likely induced by protein conformational changes during the isolation/purification procedures. Absorption, emission, and HB spectra of WTM and L122Q mutant are very similar, in which the low-energy trap (revealed by the nonresonant HB spectra) shifts to higher energies as a function of fluence, supporting a mixture model. No fluence-dependent shift is observed in the WTI FMO trimers. New Hamiltonians are provided for WTI and WTD proteins. Resonant HB spectra show that the internal energy relaxation times in the WTM and L122Q mutant are similar, and depend on excitation frequency. Fast average relaxation times (excited state lifetimes) are observed for burning into the main broad absorption band near 805nm. Burning at longer wavelengths reveals slower total dephasing times. No resonant bleach is observed at λB≤803nm, implying much faster (femtosecond) energy relaxation in this spectral range in agreement with 2D electronic spectroscopy frequency maps.

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review.

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.