High-Resolution Frequency-Domain Spectroscopic and Modeling Studies of Photosystem I (PSI), PSI Mutants and PSI Supercomplexes.

Photosystem I (PSI) is one of the two main pigment-protein complexes where the primary steps of oxygenic photosynthesis take place. This review describes low-temperature frequency-domain experiments (absorption, emission, circular dichroism, resonant and non-resonant hole-burned spectra) and modeling efforts reported for PSI in recent years. In particular, we focus on the spectral hole-burning studies, which are not as common in photosynthesis research as the time-domain spectroscopies. Experimental and modeling data obtained for trimeric cyanobacterial Photosystem I (PSI3), PSI3 mutants, and PSI3-IsiA18 supercomplexes are analyzed to provide a more comprehensive understanding of their excitonic structure and excitation energy transfer (EET) processes. Detailed information on the excitonic structure of photosynthetic complexes is essential to determine the structure-function relationship. We will focus on the so-called "red antenna states" of cyanobacterial PSI, as these states play an important role in photochemical processes and EET pathways. The high-resolution data and modeling studies presented here provide additional information on the energetics of the lowest energy states and their chlorophyll (Chl) compositions, as well as the EET pathways and how they are altered by mutations. We present evidence that the low-energy traps observed in PSI are excitonically coupled states with significant charge-transfer (CT) character. The analysis presented for various optical spectra of PSI3 and PSI3-IsiA18 supercomplexes allowed us to make inferences about EET from the IsiA18 ring to the PSI3 core and demonstrate that the number of entry points varies between sample preparations studied by different groups. In our most recent samples, there most likely are three entry points for EET from the IsiA18 ring per the PSI core monomer, with two of these entry points likely being located next to each other. Therefore, there are nine entry points from the IsiA18 ring to the PSI3 trimer. We anticipate that the data discussed below will stimulate further research in this area, providing even more insight into the structure-based models of these important cyanobacterial photosystems.

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.

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.

Challenges facing an understanding of the nature of low-energy excited states in photosynthesis.

While the majority of the photochemical states and pathways related to the biological capture of solar energy are now well understood and provide paradigms for artificial device design, additional low-energy states have been discovered in many systems with obscure origins and significance. However, as low-energy states are naively expected to be critical to function, these observations pose important challenges. A review of known properties of low energy states covering eight photochemical systems, and options for their interpretation, are presented. A concerted experimental and theoretical research strategy is suggested and outlined, this being aimed at providing a fully comprehensive understanding.

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.

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.

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.

Probing environment fluctuations by two-dimensional electronic spectroscopy of molecular systems at temperatures below 5 K.

Two-dimensional (2D) electronic spectroscopy at cryogenic and room temperatures reveals excitation energy relaxation and transport, as well as vibrational dynamics, in molecular systems. These phenomena are related to the spectral densities of nuclear degrees of freedom, which are directly accessible by means of hole burning and fluorescence line narrowing approaches at low temperatures (few K). The 2D spectroscopy, in principle, should reveal more details about the fluctuating environment than the 1D approaches due to peak extension into extra dimension. By studying the spectral line shapes of a dimeric aggregate at low temperature, we demonstrate that 2D spectra have the potential to reveal the fluctuation spectral densities for different electronic states, the interstate correlation of static disorder and, finally, the time scales of spectral diffusion with high resolution.

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.

On destabilization of the Fenna-Matthews-Olson complex of Chlorobaculum tepidum.

The Fenna-Matthews-Olson (FMO) complex from the green sulfur bacterium Chlorobaculum tepidum was studied with respect to its stability. We provide a critical assessment of published and recently measured optical spectra. FMO complexes were found to destabilize over time producing spectral shifts, with destabilized samples having significantly higher hole-burning efficiencies; indicating a remodeled protein energy landscape. Observed correlated peak shifts near 825 and 815 nm suggest possible correlated (protein) fluctuations. It is proposed that the value of 35 cm(-1) widely used for reorganization energy (E λ ), which has important implications for the contributions to the coherence rate (Kreisbeck and Kramer 3:2828-2833, 2012), in various modeling studies of two-dimensional electronic spectra is overestimated. We demonstrate that the value of E λ is most likely about 15-22 cm(-1) and suggest that spectra reported in the literature (often measured on different FMO samples) exhibit varied peak positions due to different purification/isolation procedures or destabilization effects.

Effect of the LHCII pigment-protein complex aggregation on photovoltaic properties of sensitized TiO2 solar cells.

A modified dye-sensitized solar cell consisting of a thin TiO2 barrier layer sensitized with natural trimeric light-harvesting complex II (LHCII) from spinach was used as a biomimetic model to study the effects of LHCII aggregation on the photovoltaic properties. The aggregation of individual trimers induced molecular reorganization, which dramatically increased the photocurrent. The morphology of small- and large-size LHCII aggregates deposited on a surface was confirmed by atomic force microscopy. Enhanced LHCII immobilization was accomplished via electrostatic interaction with amine-functionalized photoanodes. The photocurrent responses of the assembled solar cells under illumination at three characteristic wavelength bands in the UV-Vis absorption spectra of LHCII solutions confirmed that a significant photocurrent was generated by LHCII photosensitizers. The enhanced photocurrent by large aggregated LHCII is shown to correlate with the quenching in the far-red fluorescence deriving from chlorophyll-chlorophyll charge transfer states that are effectively coupled with the TiO2 surface and thus inject electrons into the TiO2 conduction band. The large aggregated LHCII with more chlorophyll-chlorophyll charge transfer states is a much better sensitizer since it injects electrons more efficiently into the conduction band of TiO2 than the small aggregated LHCII mostly consisting of unquenched chlorophyll excited state. The assembled solar cells demonstrated remarkable stability in both aqueous buffer and acetonitrile electrolytes over 30 days.

Modeling of fluorescence line-narrowed spectra in weakly coupled dimers in the presence of excitation energy transfer.

This work describes simple analytical formulas to describe the fluorescence line-narrowed (FLN) spectra of weakly coupled chromophores in the presence of excitation energy transfer (EET). Modeling studies for dimer systems (assuming low fluence and weak coupling) show that the FLN spectra (including absorption and emission spectra) calculated for various dimers using our model are in good agreement with spectra calculated by: (i) the simple convolution method and (ii) the more rigorous treatment using the Redfield approach [T. Renger and R. A. Marcus, J. Chem. Phys. 116, 9997 (2002)]. The calculated FLN spectra in the presence of EET of all three approaches are very similar. We argue that our approach provides a simplified and computationally more efficient description of FLN spectra in the presence of EET. This method also has been applied to FLN spectra obtained for the CP47 antenna complex of Photosystem II reported by Neupane et al. [J. Am. Chem. Soc. 132, 4214 (2010)], which indicated the presence of uncorrelated EET between pigments contributing to the two lowest energy (overlapping) exciton states, each mostly localized on a single chromophore. Calculated and experimental FLN spectra for CP47 complex show very good qualitative agreement.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

Direct synthesis of aqueous quantum dots through 4,4'-bipyridine-based twin ligand strategy.

We report a new class of derivatized 4,4'-bipyridinium ligands for use in synthesizing highly fluorescent, extremely stable, water-soluble CdSe and CdTe quantum dots (QDs) for bioconjugation. We employed an evaporation-condensation technique, also known as solvated metal atom dispersion (SMAD), followed by a digestive ripening procedure. This method has been used to synthesize both metal nanoparticles and semiconductors in the gram scale with several stabilizing ligands in various solvents. The SMAD technique comprised evaporation condensation and stabilization of CdSe or CdTe in tetrahydrofuran. The as-prepared product was then digestively ripened in both water and dimethyl formamide, leading to narrowing of the particle size distributions. The ligands were synthesized by nucleophilic substitution (S(N)2) reactions using 4,4'-bipyridine as a nucleophile. Confocal microscopy images revealed the orange color of the nanocrystalline QDs with diameters of ~5 nm. The size has been confirmed by using transmission electron microscopy. As a part of our strategy, 85% of the 4,4'-bipyridinium salt was synthesized as propionic acid derivative and used to both stabilize the QDs in water and label basic amino acids and different biomarkers utilizing the carboxylic acid functional group. Fifteen percent of the 4,4'-bipyridinium salt was synthesized as N-propyl maleimide and used as a second ligand to label any protein containing the amino acid cysteine by means of a 1,4-Michael addition.

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.

Integrated microfluidic device for the separation and electrochemical detection of catechol estrogen-derived DNA adducts.

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4-10 µM with r(2) ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.

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.

Insight into the electronic structure of the CP47 antenna protein complex of photosystem II: hole burning and fluorescence study.

We report low temperature (T) optical spectra of the isolated CP47 antenna complex from Photosystem II (PSII) with a low-T fluorescence emission maximum near 695 nm and not, as previously reported, at 690-693 nm. The latter emission is suggested to result from three distinct bands: a lowest-state emission band near 695 nm (labeled F1) originating from the lowest-energy excitonic state A1 of intact complexes (located near 693 nm and characterized by very weak oscillator strength) as well as emission peaks near 691 nm (FT1) and 685 nm (FT2) originating from subpopulations of partly destabilized complexes. The observation of the F1 emission is in excellent agreement with the 695 nm emission observed in intact PSII cores and thylakoid membranes. We argue that the band near 684 nm previously observed in singlet-minus-triplet spectra originates from a subpopulation of partially destabilized complexes with lowest-energy traps located near 684 nm in absorption (referred to as AT2) giving rise to FT2 emission. It is demonstrated that varying contributions from the F1, FT1, and FT2 emission bands led to different maxima of fluorescence spectra reported in the literature. The fluorescence spectra are consistent with the zero-phonon hole action spectra obtained in absorption mode, the profiles of the nonresonantly burned holes as a function of fluence, as well as the fluorescence line-narrowed spectra obtained for the Q(y) band. The lowest Q(y) state in absorption band (A1) is characterized by an electron-phonon coupling with the Huang-Rhys factor S of approximately 1 and an inhomogeneous width of approximately 180 cm(-1). The mean phonon frequency of the A1 band is 20 cm(-1). In contrast to previous observations, intact isolated CP47 reveals negligible contribution from the triplet-bottleneck hole, i.e., the AT2 trap. It has been shown that Chls in intact CP47 are connected via efficient excitation energy transfer to the A1 trap near 693 nm and that the position of the fluorescence maximum depends on the burn fluence. That is, the 695 nm fluorescence maximum shifts blue with increasing fluence, in agreement with nonresonant hole burned spectra. The above findings provide important constraints and parameters for future excitonic calculations, which in turn should offer new insight into the excitonic structure and composition of low-energy absorption traps.

Accurate modeling of fluorescence line narrowing difference spectra: Direct measurement of the single-site fluorescence spectrum.

Accurate lineshape functions for modeling fluorescence line narrowing (FLN) difference spectra (DeltaFLN spectra) in the low-fluence limit are derived and examined in terms of the physical interpretation of various contributions, including photoproduct absorption and emission. While in agreement with the earlier results of Jaaniso [Proc. Est. Acad. Sci., Phys., Math. 34, 277 (1985)] and Funfschilling et al. [J. Lumin. 36, 85 (1986)], the derived formulas differ substantially from functions used recently [e.g., M. Ratsep et al., Chem. Phys. Lett. 479, 140 (2009)] to model DeltaFLN spectra. In contrast to traditional FLN spectra, it is demonstrated that for most physically reasonable parameters, the DeltaFLN spectrum reduces simply to the single-site fluorescence lineshape function. These results imply that direct measurement of a bulk-averaged single-site fluorescence lineshape function can be accomplished with no complicated extraction process or knowledge of any additional parameters such as site distribution function shape and width. We argue that previous analysis of DeltaFLN spectra obtained for many photosynthetic complexes led to strong artificial lowering of apparent electron-phonon coupling strength, especially on the high-energy side of the pigment site distribution function.