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.

On wavelength-dependent exciton lifetime distributions in reconstituted CP29 antenna of the photosystem II and its site-directed mutants.

To provide more insight into the excitonic structure and exciton lifetimes of the wild type (WT) CP29 complex of photosystem II, we measured high-resolution (low temperature) absorption, emission, and hole burned spectra for the A2 and B3 mutants, which lack chlorophylls a612 and b614 (Chls), respectively. Experimental and modeling results obtained for the WT CP29 and A2/B3 mutants provide new insight on the mutation-induced changes at the molecular level and shed more light on energy transfer dynamics. Simulations of the A2 and B3 optical spectra, using the second-order non-Markovian theory, and comparison with improved fits of WT CP29 optical spectra provide more insight into their excitonic structure, mutation induced changes, and frequency-dependent distributions of exciton lifetimes (T1). A new Hamiltonian obtained for WT CP29 reveals that deletion of Chls a612 or b614 induces changes in the site energies of all remaining Chls. Hamiltonians obtained for A2 and B3 mutants are discussed in the context of the energy landscape of chlorophylls, excitonic structure, and transfer kinetics. Our data suggest that the lowest exciton states in A2 and B3 mutants are contributed by a611(57%), a610(17%), a615(15%) and a615(58%), a611(20%), a612(15%) Chls, respectively, although other compositions of lowest energy states are also discussed. Finally, we argue that the calculated exciton decay times are consistent with both the hole-burning and recent transient absorption measurements. Wavelength-dependent T1 distributions offer more insight into the interpretation of kinetic traces commonly described by discrete exponentials in global analysis/global fitting of transient absorption experiments.

On Excitation Energy Transfer within the Baseplate BChl a-CsmA Complex of Chloroflexus aurantiacus.

Recently, a hybrid approach combining solid-state NMR spectroscopy and cryo-electron microscopy showed that the baseplate in green sulfur bacterium Chlorobaculum tepidum is a 2D lattice of BChl a-CsmA dimers [Nielsen, J. T.; et al., Nat. Commun. 2016, 7, 12454-12465]. While the existence of the BChl a-CsmA subunit was previously known, the proposed orientations of the BChl a pigments had only been elucidated from spectral data up to this point. Regarding the electronic structure of the baseplate, two models have been proposed. 2D electronic spectroscopy data were interpreted as revealing that at least four excitonically coupled BChl a might be in close contact. Conversely, spectral hole burning data suggested that the lowest energy state was localized, yet additional states are sometimes observed because of the presence of the Fenna-Matthews-Olson (FMO) antenna protein. To solve this conundrum, this work studies the chlorosome-baseplate complex from Chloroflexus aurantiacus, which does not contain the FMO protein. The results confirm that in both C. tepidum and C. aurantiacus, excitation energy is transferred to a localized low-energy trap state near 818 nm with similar rates, most likely via exciton hopping.

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.

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).

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.

On Light-Induced Photoconversion of B800 Bacteriochlorophylls in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum.

The B800-850 LH2 antenna from the photosynthetic purple sulfur bacterium Allochromatium vinosum exhibits an unusual spectral splitting of the B800 absorption band; i.e., two bands are well-resolved at 5 K with maxima at 805 nm (B800R) and 792 nm (B800B). To provide more insight into the nature of the B800 bacteriochlorophyll (BChl) a molecules, high-resolution hole-burning (HB) spectroscopy is employed. Both white light illumination and selective laser excitations into B800R or B800B lead to B800R → B800B phototransformation. Selective excitation into B800B leads to uncorrelated excitation energy transfer (EET) to B800R and subsequent B800R → B800B phototransformation. The B800B → B800R EET time is 0.9 ± 0.1 ps. Excitation at 808.4 nm (into the low-energy side of B800R) shows that the lower limit of B800R → B850 EET is about 2 ps, as the B800R → B800B phototransformation process could contribute to the corresponding zero-phonon hole width. The phototransformation of B800R leads to a ∼ 200 cm-1 average blue-shift of transition energies, i.e., B800R changes into B800B. We argue that it is unlikely that B800-B850 excitonic interactions give rise to a splitting of the B800 band. We propose that the latter is caused by different protein conformations that can lead to both strong or weak hydrogen bond(s) between B800 pigments and the protein scaffolding. Temperature-dependent absorption spectra of B800, which revealed a well-defined isosbestic point, support a two-site model, likely with strongly and weakly hydrogen-bonded B800 BChls. Thus, BChls contributing to B800R and B800B could differ in the position of the proton in the BChl carbonyl-protein hydrogen bond, i.e., proton dynamics along the hydrogen bond may well be the major mechanism of this phototransformation. However, the effective tunneling mass is likely larger than the proton mass.

Polymer-Chlorosome Nanocomposites Consisting of Non-Native Combinations of Self-Assembling Bacteriochlorophylls.

Chlorosomes are one of the characteristic light-harvesting antennas from green sulfur bacteria. These complexes represent a unique paradigm: self-assembly of bacteriochlorophyll pigments within a lipid monolayer without the influence of protein. Because of their large size and reduced complexity, they have been targeted as models for the development of bioinspired light-harvesting arrays. We report the production of biohybrid light-harvesting nanocomposites mimicking chlorosomes, composed of amphiphilic diblock copolymer membrane bodies that incorporate thousands of natural self-assembling bacteriochlorophyll molecules derived from green sulfur bacteria. The driving force behind the assembly of these polymer-chlorosome nanocomposites is the transfer of the mixed raw materials from the organic to the aqueous phase. We incorporated up to five different self-assembling pigment types into single nanocomposites that mimic chlorosome morphology. We establish that the copolymer-BChl self-assembly process works smoothly even when non-native combinations of BChl homologues are included. Spectroscopic characterization revealed that the different types of self-assembling pigments participate in ultrafast energy transfer, expanding beyond single chromophore constraints of the natural chlorosome system. This study further demonstrates the utility of flexible short-chain, diblock copolymers for building scalable, tunable light-harvesting arrays for technological use and allows for an in vitro analysis of the flexibility of natural self-assembling chromophores in unique and controlled combinations.

Conformational Complexity in the LH2 Antenna of the Purple Sulfur Bacterium Allochromatium vinosum Revealed by Hole-Burning Spectroscopy.

This work discusses the protein conformational complexity of the B800-850 LH2 complexes from the purple sulfur bacterium Allochromatium vinosum, focusing on the spectral characteristics of the B850 chromophores. Low-temperature B850 absorption and the split B800 band shift blue and red, respectively, at elevated temperatures, revealing isosbestic points. The latter indicates the presence of two (unresolved) conformations of B850 bacteriochlorophylls (BChls), referred to as conformations 1 and 2, and two conformations of B800 BChls, denoted as B800R and B800B. The energy differences between average site energies of conformations 1 and 2, and B800R and B800B are similar (∼200 cm-1), suggesting weak and strong hydrogen bonds linking two major subpopulations of BChls and the protein scaffolding. Although conformations 1 and 2 of the B850 chromophores, and B800R and B800B, exist in the ground state, selective excitation leads to 1 → 2 and B800R → B800B phototransformations. Different static inhomogeneous broadening is revealed for the lowest energy exciton states of B850 (fwhm ∼195 cm-1) and B800R (fwhm ∼140 cm-1). To describe the 5 K absorption spectrum and the above-mentioned conformations, we employ an exciton model with dichotomous protein conformation disorder. We show that both experimental data and the modeling study support a two-site model with strongly and weakly hydrogen-bonded B850 and B800 BChls, which under illumination undergo conformational changes, most likely caused by proton dynamics.

Toward an Understanding of the Excitonic Structure of the CP47 Antenna Protein Complex of Photosystem II Revealed via Circularly Polarized Luminescence.

Identification of the lowest energy pigments in the photosynthetic CP47 antenna protein complex of Photosystem II (PSII) is essential for understanding its excitonic structure, as well as excitation energy pathways in the PSII core complex. Unfortunately, there is no consensus concerning the nature of the low-energy state(s), nor chlorophyll (Chl) site energies in this important photosynthetic antenna. Although we raised concerns regarding the estimations of Chl site energies obtained from modeling studies of various types of CP47 optical spectra [Reinot, T; et al., Anal. Chem. Insights 2016, 11, 35-48] recent new assignments imposed by the shape of the circularly polarized luminescence (CPL) spectrum [Hall, J.; et al., Biochim. Biophys. Acta 2016, 1857, 1580-1593] necessitate our comments. We demonstrate that other combinations of low-energy Chls provide equally good or improved simultaneous fits of various optical spectra (absorption, emission, CPL, circular dichroism, and nonresonant hole-burned spectra), but more importantly, we expose the heterogeneous nature of the recently studied complexes and argue that the published composite nature of the CPL (contributed to by CPL685, CPL691, and CPL695) does not represent an intact CP47 protein. A positive CPL695 is extracted for the intact protein, which, when simultaneously fitted with multiple other optical spectra, provides new information on the excitonic structure of intact and destabilized CP47 complexes and their lowest energy state(s).

Effect of Spectral Density Shapes on the Excitonic Structure and Dynamics of the Fenna-Matthews-Olson Trimer from Chlorobaculum tepidum.

The Fenna-Matthews-Olson (FMO) trimer (composed of identical subunits) from the green sulfur bacterium Chlorobaculum tepidum is an important protein model system to study exciton dynamics and excitation energy transfer (EET) in photosynthetic complexes. In addition, FMO is a popular model for excitonic calculations, with many theoretical parameter sets reported describing different linear and nonlinear optical spectra. Due to fast exciton relaxation within each subunit, intermonomer EET results predominantly from the lowest energy exciton states (contributed to by BChl a 3 and 4). Using experimentally determined shapes for the spectral densities, simulated optical spectra are obtained for the entire FMO trimer. Simultaneous fits of low-temperature absorption, fluorescence, and hole-burned spectra place constraints on the determined pigment site energies, providing a new Hamiltonian that should be further tested to improve modeling of 2D electronic spectroscopy data and our understanding of coherent and dissipation effects in this important protein complex.

Mutation-Induced Changes in the Protein Environment and Site Energies in the (M)L214G Mutant of the Rhodobacter sphaeroides Bacterial Reaction Center.

This work focuses on the low-temperature (5 K) photochemical (transient) hole-burned (HB) spectra within the P870 absorption band, and their theoretical analysis, for the (M)L214G mutant of the photosynthetic Rhodobacter sphaeroides bacterial reaction center (bRC). To provide insight into system-bath interactions of the bacteriochlorophyll a (BChl a) special pair, i.e., P870, in the mutated bRC, the optical line shape function for the P870 band is calculated numerically. On the basis of the modeling studies, we demonstrate that (M)L214G mutation leads to a heterogeneous population of bRCs with modified (increased) total electron-phonon coupling strength of the special pair BChl a and larger inhomogeneous broadening. Specifically, we show that after mutation in the (M)L214G bRC a large fraction (∼50%) of the bacteriopheophytin (HA) chromophores shifts red and the 800 nm absorption band broadens, while the remaining fraction of HA cofactors retains nearly the same site energy as HA in the wild-type bRC. Modeling using these two subpopulations allowed for fits of the absorption and nonresonant (transient) HB spectra of the mutant bRC in the charge neutral, oxidized, and charge-separated states using the Frenkel exciton Hamiltonian, providing new insight into the mutant's complex electronic structure. Although the average (M)L214G mutant quantum efficiency of P(+)QA(-) state formation seems to be altered in comparison with the wild-type bRC, the average electron transfer time (measured via resonant transient HB spectra within the P870 band) was not affected. Thus, mutation in the vicinity of the electron acceptor (HA) does not tune the charge separation dynamics. Finally, quenching of the (M)L214G mutant excited states by P(+) is addressed by persistent HB spectra burned within the B band in chemically oxidized samples.

On the Conflicting Estimations of Pigment Site Energies in Photosynthetic Complexes: A Case Study of the CP47 Complex.

We focus on problems with elucidation of site energies [Formula: see text] for photosynthetic complexes (PSCs) in order to raise some genuine concern regarding the conflicting estimations propagating in the literature. As an example, we provide a stern assessment of the site energies extracted from fits to optical spectra of the widely studied CP47 antenna complex of photosystem II from spinach, though many general comments apply to other PSCs as well. Correct values of [Formula: see text] for chlorophyll (Chl) a in CP47 are essential for understanding its excitonic structure, population dynamics, and excitation energy pathway(s). To demonstrate this, we present a case study where simultaneous fits of multiple spectra (absorption, emission, circular dichroism, and nonresonant hole-burned spectra) show that several sets of parameters can fit the spectra very well. Importantly, we show that variable emission maxima (690-695 nm) and sample-dependent bleaching in nonresonant hole-burning spectra reported in literature could be explained, assuming that many previously studied CP47 samples were a mixture of intact and destabilized proteins. It appears that the destabilized subpopulation of CP47 complexes could feature a weakened hydrogen bond between the 13(1)-keto group of Chl29 and the PsbH protein subunit, though other possibilities cannot be entirely excluded, as discussed in this work. Possible implications of our findings are briefly discussed.

Band Structure of the Rhodobacter sphaeroides Photosynthetic Reaction Center from Low-Temperature Absorption and Hole-Burned Spectra.

Persistent/transient spectral hole burning (HB) and computer simulations are used to provide new insight into the excitonic structure and excitation energy transfer of the widely studied bacterial reaction center (bRC) of Rhodobacter (Rb.) sphaeroides. We focus on site energies of its cofactors and electrochromic shifts induced in the chemically oxidized (P(+)) and charge-separated (P(+)QM(-)) states. Theoretical models lead to two alternative interpretations of the H-band. On the basis of our experimental and simulation data, we suggest that the bleach near 813-825 nm in transient HB spectra in the P(+)QM(-) state, often assigned to the upper exciton component of the special pair, is mostly due to different electrochromic shifts of the BL/M cofactors. From the exciton compositions in the charge-neutral (CN) bRC, the weak fourth excitonic band near 780 nm can be denoted PY+, that is, the upper excitonic band of the special pair, which in the CN bRC behaves as a delocalized state over PM and PL pigments that weakly mixes with accessory BChls. Thus, the shoulder in the absorption of Rb. sphaeroides near 813-815 nm does not contain the PY+ exciton band.

New Insight into the Water-Soluble Chlorophyll-Binding Protein from Lepidium virginicum.

This study describes new recombinant water-soluble chlorophyll (Chl)-binding proteins (WSCP) from Lepidium virginicum (LvWSCP). This complex binds four Chls (i.e. two dimers of Chls) per protein tetramer. We show that absorption, emission, hole-burned (HB) spectra and the shape of the zero-phonon hole (ZPH) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.-J. et al. (2008) J. Phys. Chem. B, 112, 13951; Pieper, J. et al. (2011) J. Phys. Chem. B, 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e. electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra (i.e. the absence of antihole), demonstrating that the hole-burning process in LvWSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.

Alternative Excitonic Structure in the Baseplate (BChl a-CsmA Complex) of the Chlorosome from Chlorobaculum tepidum.

In the photosynthetic green sulfur bacterium Chlorobaculum tepidum, the baseplate mediates excitation energy transfer from the light-harvesting chlorosome to the Fenna-Matthews-Olson (FMO) complex and subsequently toward the reaction center (RC). Literature data suggest that the baseplate is a 2D lattice of BChl a-CsmA dimers. However, recently, it has been proposed, using 2D electronic spectroscopy (2DES) at 77 K, that at least four excitonically coupled BChl a are in close contact within the baseplate structure [ Dostál , J. ; et al., J. Phys. Chem. Lett. 2014 , 5 , 1743 ]. This finding is tested via hole burning (HB) spectroscopy (5 K). Our results indicate that the four excitonic states identified by 2DES likely correspond to contamination of the baseplate with the FMO antenna and possibly the RC. In contrast, HB reveals a different excitonic structure of the baseplate chromophores, where excitation is transferred to a localized trap state near 818 nm via exciton hopping, which leads to emission near 826 nm.

Critical assessment of the emission spectra of various photosystem II core complexes.

We evaluate low-temperature (low-T) emission spectra of photosystem II core complexes (PSII-cc) previously reported in the literature, which are compared with emission spectra of PSII-cc obtained in this work from spinach and for dissolved PSII crystals from Thermosynechococcus (T.) elongatus. This new spectral dataset is used to interpret data published on membrane PSII (PSII-m) fragments from spinach and Chlamydomonas reinhardtii, as well as PSII-cc from T. vulcanus and intentionally damaged PSII-cc from spinach. This study offers new insight into the assignment of emission spectra reported on PSII-cc from different organisms. Previously reported spectra are also compared with data obtained at different saturation levels of the lowest energy state(s) of spinach and T. elongatus PSII-cc via hole burning in order to provide more insight into emission from bleached and/or photodamaged complexes. We show that typical low-T emission spectra of PSII-cc (with closed RCs), in addition to the 695 nm fluorescence band assigned to the intact CP47 complex (Reppert et al. J Phys Chem B 114:11884-11898, 2010), can be contributed to by several emission bands, depending on sample quality. Possible contributions include (i) a band near 690-691 nm that is largely reversible upon temperature annealing, proving that the band originates from CP47 with a bleached low-energy state near 693 nm (Neupane et al. J Am Chem Soc 132:4214-4229, 2010; Reppert et al. J Phys Chem B 114:11884-11898, 2010); (ii) CP43 emission at 683.3 nm (not at 685 nm, i.e., the F685 band, as reported in the literature) (Dang et al. J Phys Chem B 112:9921-9933, 2008; Reppert et al. J Phys Chem B 112:9934-9947, 2008); (iii) trap emission from destabilized CP47 complexes near 691 nm (FT1) and 685 nm (FT2) (Neupane et al. J Am Chem Soc 132:4214-4229, 2010); and (iv) emission from the RC pigments near 686-687 nm. We suggest that recently reported emission of single PSII-cc complexes from T. elongatus may not represent intact complexes, while those obtained for T. elongatus presented in this work most likely represent intact PSII-cc, since they are nearly indistinguishable from emission spectra obtained for various PSII-m fragments.

Comments on the optical lineshape function: application to transient hole-burned spectra of bacterial reaction centers.

The vibrational spectral density is an important physical parameter needed to describe both linear and non-linear spectra of multi-chromophore systems such as photosynthetic complexes. Low-temperature techniques such as hole burning (HB) and fluorescence line narrowing are commonly used to extract the spectral density for a given electronic transition from experimental data. We report here that the lineshape function formula reported by Hayes et al. [J. Phys. Chem. 98, 7337 (1994)] in the mean-phonon approximation and frequently applied to analyzing HB data contains inconsistencies in notation, leading to essentially incorrect expressions in cases of moderate and strong electron-phonon (el-ph) coupling strengths. A corrected lineshape function L(ω) is given that retains the computational and intuitive advantages of the expression of Hayes et al. [J. Phys. Chem. 98, 7337 (1994)]. Although the corrected lineshape function could be used in modeling studies of various optical spectra, we suggest that it is better to calculate the lineshape function numerically, without introducing the mean-phonon approximation. New theoretical fits of the P870 and P960 absorption bands and frequency-dependent resonant HB spectra of Rb. sphaeroides and Rps. viridis reaction centers are provided as examples to demonstrate the importance of correct lineshape expressions. Comparison with the previously determined el-ph coupling parameters [Johnson et al., J. Phys. Chem. 94, 5849 (1990); Lyle et al., ibid. 97, 6924 (1993); Reddy et al., ibid. 97, 6934 (1993)] is also provided. The new fits lead to modified el-ph coupling strengths and different frequencies of the special pair marker mode, ωsp, for Rb. sphaeroides that could be used in the future for more advanced calculations of absorption and HB spectra obtained for various bacterial reaction centers.

Does the singlet minus triplet spectrum with major photobleaching band near 680-682 nm represent an intact reaction center of Photosystem II?

We use both frequency- and time-domain low-temperature (5-20 K) spectroscopies to further elucidate the shape and spectral position of singlet minus triplet (triplet-bottleneck) spectra in the reaction centers (RCs) of Photosystem II (PSII) isolated from wild-type Chlamydomonas reinhardtii and spinach. It is shown that the shape of the nonresonant transient hole-burned spectrum in destabilized RCs from C. reinhardtii is very similar to that typically observed for spinach. This suggests that the previously observed difference in transient spectra between RCs from C. reinhardtii and spinach is not due to the sample origin but most likely due to a partial destabilization of the D1 and D2 polypeptides. This supports our previous assignments that destabilized RCs (referred to as RC680) (Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870), with a major photobleaching band near 680-682 nm and the absence of a photobleaching band near 673 nm, do not represent the intact RC residing within the PSII core complex. Time-resolved absorption difference spectra obtained for partially destabilized RCs of C. reinhardtii and for typical spinach RCs support the above conclusions. The absence of clear photobleaching bands near 673 and 684 nm (where the PD1 chlorophyll and the active pheophytin (PheoD1) contribute, respectively) in picosecond transient absorption spectra in both RCs studied in this work indicates that the cation can move from the primary electron donor (ChlD1) to PD1 (i.e., PD1ChlD1(+)PheoD1(-) → PD1(+)ChlD1PheoD1(-)). Therefore, we suggest that ChlD1 is the major electron donor in usually studied destabilized RCs (with a major photobleaching near 680-682 nm), although the PD1 path (where PD1 serves as the primary electron donor) is likely present in intact RCs, as discussed in Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870.