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.

Bound detergent molecules in bacterial reaction centers facilitate detection of tetryl explosive.

Bacterial reaction centers (BRC) from Rhodobacter sphaeroides were found to accelerate, about 100-fold, the reaction between tetryl (2,4,6-trinitrophenylmethylnitramine) explosive and n-lauryl-N-N-dimethylamine-N-oxide (LDAO) that results in the formation of picric acid-like product with characteristic UV-VIS absorption spectrum with peaks at 345 and 415 nm. Moreover, this product also affects the spectra of BRC cofactors in the NIR spectral region and stabilizes the conformational changes associated with slow charge recombination. The evolution of the NIR absorption changes correlated with the kinetics of the product formation. Comparison between the wild-type and the R26 carotenoid-less strain indicates that tetryl-LDAO reaction is roughly five times faster for R26, which allows for identifying the carotenoid binding site as the optimal reaction site. Another, less-defined reaction site is located in the BRC's hydrophobic cavity. These effects are highly selective for tetryl and not observed for several other widespread nitric explosives; slowed-down charge recombination allows for distinguishing between tetryl and QB-site herbicides. The current limit of detection is in the ppb range or ~ 100 nM. Details of the molecular mechanisms of the reactions and perspectives of using these effects in bioassays or biosensors for explosives detection are also discussed.

A simple and efficient method to prepare pure dimers and monomers of the cytochrome b 6 f complex from spinach.

Using a single size-exclusion chromatography we were able to isolate highly pure dimers and monomers of the Cyt b 6 f complex from spinach from a bulk preparation of that protein complex obtained with a standard procedure. At higher protein/detergent ratio during the chromatography most of the Cyt b 6 f complex remained as dimers. In contrast, at lower protein/detergent ratio (around 15 times lower), most dimers became monomerized. As a bonus, this chromatography also allowed the elimination of potential Chl a contaminant to the Cyt b 6 f preparations. SDS-PAGE protein analysis with 18% (w/v) acrylamide revealed the loss of the ISP subunit in our monomeric preparation. However, it fully retained the content of Chl a, a prerequisite to perform any spectroscopic study involving this unique pigment.

Identification of Residues Potentially Involved in Optical Shifts in the Water-Soluble Chlorophyll a-Binding Protein through Molecular Dynamics Simulations.

Reversible light and thermally induced spectral shifts are universally observed in a wide variety of pigment-protein complexes at temperatures ranging from cryogenic to ambient. In this paper, we employed large-scale molecular dynamics (MD) simulations of a prototypical pigment-protein complex to better understand these shifts at a molecular scale. Although multiple mechanisms have been proposed over the years, no verification of these proposals via MD simulations has thus far been performed; our work represents the first step in this direction. From simulations of the water-soluble chlorophyll-binding protein complex, we determined that rearrangements of long hydrogen bonds were unlikely to be the origin of the multiwell landscape features necessary to explain observed spectral shifts. We also assessed small motions of amino acid residues and identified side chain rotations of some of these residues as likely candidates for the origin of relevant multiwell landscape features. The protein free-energy landscapes associated with side chain rotations feature energy barriers of around 1100-1600 cm-1, in agreement with optical spectroscopy results, with the most promising residue type associated with experimental signatures being serine, which possesses a symmetric triple-well landscape and moment of inertia of a relevant magnitude.

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.

Effects of Chlorophyll Triplet States on the Kinetics of Spectral Hole Growth.

Spectral hole burning has been employed for decades to study various amorphous solids and proteins. Triplet states and respective transient holes were incorporated into theoretical models and software simulating nonphotochemical spectral hole burning (NPHB) and including all relevant distributions, in particular the distribution of the angle between the electric field of light E and transient dipole moment of the chromophore µ. The presence of a chlorophyll a triplet state with a lifetime of several milliseconds explains the slowdown of NPHB (on the depth vs illumination dose scale) with the increase of the light intensity, as well as larger hole depths observed in weak probe beam experiments, compared to those deduced from the hole growth kinetics (HGK) measurements (signal collected at a fixed wavelength while a stronger burning beam is on) in cytochrome b6f and chemically modified LH2. We also considered the solvent deuteration effects on triplet lifetime and concluded that both triplet states and local heating likely play a role in slowing down the HGK with increasing burn intensity.

Functional analysis of low-grade glioma genetic variants predicts key target genes and transcription factors.

BACKGROUND: Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. METHODS: We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained on TF binding data, and simulated annealing-based interpretation methods. RESULTS: We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. CONCLUSIONS: The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.

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.

How Well Does the Hole-Burning Action Spectrum Represent the Site-Distribution Function of the Lowest-Energy State in Photosynthetic Pigment-Protein Complexes?

For the first time, we combined Monte Carlo and nonphotochemical hole burning (NPHB) master equation approaches to allow for ultrahigh-resolution (<0.005 cm-1, smaller than the typical homogeneous line widths at 5 K) simulations of the NPHB spectra of dimers and trimers of interacting pigments. These simulations reveal significant differences between the zero-phonon hole (ZPH) action spectrum and the site-distribution function (SDF) of the lowest-energy state. The NPHB of the lowest-energy pigment, following the excitation energy transfer (EET) from the higher-energy pigments which are excited directly, results in the shifts of all excited states. These shifts affect the ZPH action spectra and EET times derived from the widths of the spectral holes burned in the donor-dominated regions. The effect is present for a broad variety of realistic antihole functions, and it is maximal at relatively low values of interpigment coupling (V ≤ 5 cm-1) where the use of the Förster approximation is justified. These findings need to be considered in interpreting various optical spectra of photosynthetic pigment-protein complexes for which SDFs (describing the inhomogeneous broadening) are often obtained directly from the ZPH action spectra. Water-soluble chlorophyll-binding protein (WSCP) was considered as an example.

Evidence of Simultaneous Spectral Hole Burning Involving Two Tiers of the Protein Energy Landscape in Cytochrome b6f.

Cytochrome b6f, with one chlorophyll molecule per protein monomer, is a simple model system whose studies can help achieve a better understanding of nonphotochemical spectral hole burning (NPHB) and single-complex spectroscopy results obtained in more complicated photosynthetic chlorophyll-protein complexes. We are reporting new data and proposing an alternative explanation for spectral dynamics that was recently observed in cytochrome b6f using NPHB. The relevant distribution of the tunneling parameter λ is a superposition of two components that are nearly degenerate in terms of the resultant NPHB yield and represent two tiers of the energy landscape responsible for NPHB. These two components likely burn competitively; we present the first demonstration of modeling a competitive NPHB process. Similar values of the NPHB yield result from distinctly different combinations of barrier heights, shifts along the generalized coordinate d, and/or masses of the entities involved in conformational changes m, with md2 parameter different by a factor of 2.7. Consequently, in cytochrome b6f, the first (at least) 10 h of fixed-temperature recovery preferentially probe different components of the barrier- and λ-distributions encoded into the spectral holes than thermocycling experiments. Both components most likely represent dynamics of the protein and not of the surrounding buffer/glycerol glass.

Low-energy chlorophyll states in the CP43 antenna protein complex: simulation of various optical spectra. II.

The CP43 protein complex of the core antenna of higher plant photosystem II (PSII) has two quasidegenerate "red" absorption states. It has been shown in the accompanying paper I (Dang, N. C., et al. J. Phys. Chem. B 2008, 112, 9921.) that the site distribution functions (SDFs) of red-states A and B are uncorrelated and the narrow holes are burned in subpopulations of chlorophylls (Chls) from states A and B that are the lowest-energy pigments in their particular CP43 complexes and cannot further transfer energy downhill. In this work, we present the results of a series of Monte Carlo simulations using the 3.0-A structure of the PSII core complex from cyanobacteria (Loll, B., et al. Nature 2005, 303, 1040.) to model absorption, emission, persistent, and transient hole burned (HB) spectra. At the current structural resolution, we found calculated site energies (obtained from INDO/S calculations) to be only suggestive because their values are different for the two monomers of CP43 in the PS II dimer. As a result, to probe the excitonic structure, a simple fitting procedure was employed to optimize Chl site energies from various starting values corresponding to different A/B pigment combinations to provide simultaneously good fits to several types of optical spectra. It is demonstrated that the shape of the calculated absorption, emission, and transient/persistent hole-burned spectra is consistent with experimental data and our model for excitation energy transfer between two quasi-degenerate lowest-E states (A and B) with uncorrelated SDFs discussed in paper I. Calculations revealed that absorption changes observed near 670 nm in the non-line-narrowed persistent HB spectra (assigned to photoconversion involving Chl-protein hydrogen-bonding by Hughes (Biochemistry 2006, 45, 12345.) are most likely the result of nonphotochemical hole-burning (NPHB) accompanied by the redistribution of oscillator strength due to modified excitonic interactions. We argue that a unique redistribution of oscillator strength during the NPHB process helps to assign Chls contributing to the low-energy states. It is demonstrated that the 4.2 K asymmetric triplet-bottleneck (transient) hole is mostly contributed to by both A and B states, with the hole profile described by a subensemble of pigments, which are the lowest-energy pigments (B s- and A s-type) in their complexes. The same lowest-energy Chls contribute to the observed fluorescence spectra. On the basis of our excitonic calculations, the best Chl candidates that contribute to the low-energy A and B states are Chl 44 and Chl 37, respectively.

The CP43 proximal antenna complex of higher plant photosystem II revisited: modeling and hole burning study. I.

The CP43 core antenna complex of photosystem II is known to possess two quasi-degenerate "red"-trap states (Jankowiak, R. et al. J. Phys. Chem. B 2000, 104, 11805). It has been suggested recently (Zazubovich, V.; Jankowiak, R. J. Lumin. 2007, 127, 245) that the site distribution functions of the red states (A and B) are uncorrelated and that narrow holes are burned in the subpopulations of chlorophylls (Chls) from states A and B that are the lowest-energy Chl in their complex and previously thought not to transfer energy. This model of uncorrelated excitation energy transfer (EET) between the quasidegenerate bands is expanded by taking into account both electron-phonon and vibrational coupling. The model is applied to fit simultaneously absorption, emission, zero-phonon action, and transient hole burned (HB) spectra obtained for the CP43 complex with minimized contribution from aggregation. It is demonstrated that the above listed spectra can be well-fitted using the uncorrelated EET model, providing strong evidence for the existence of efficient energy transfer between the two lowest energy states, A and B (either from A to B or from B to A), in CP43. Possible candidate Chls for the low-energy A and B states are discussed, providing a link between CP43 structure and spectroscopy. Finally, we propose that persistent holes originate from regular NPHB accompanied by the redistribution of oscillator strength due to excitonic interactions, rather than photoconversion involving Chl-protein hydrogen bonding, as suggested before ( Hughes J. L. et al. Biochemistry 2006, 45, 12345 ). In the accompanying paper (Reppert, M.; Zazubovich, V.; Dang, N. C.; Seibert, M.; Jankowiak, R. J. Phys. Chem. B 2008, 9934), it is demonstrated that the model discussed in this manuscript is consistent with excitonic calculations, which also provide very good fits to both transient and persistent HB spectra obtained under non-line-narrowing conditions.

Red antenna states of photosystem I from cyanobacteria Synechocystis PCC 6803 and Thermosynechococcus elongatus: single-complex spectroscopy and spectral hole-burning study.

Hole-burning and single photosynthetic complex spectroscopy were used to study the excitonic structure and excitation energy-transfer processes of cyanobacterial trimeric Photosystem I (PS I) complexes from Synechocystis PCC 6803 and Thermosynechococcus elongatus at low temperatures. It was shown that individual PS I complexes of Synechocystis PCC 6803 (which have two red antenna states, i.e., C706 and C714) reveal only a broad structureless fluorescence band with a maximum near 720 nm, indicating strong electron-phonon coupling for the lowest energy C714 red state. The absence of zero-phonon lines (ZPLs) belonging to the C706 red state in the emission spectra of individual PS I complexes from Synechocystis PCC 6803 suggests that the C706 and C714 red antenna states of Synechocystis PCC 6803 are connected by efficient energy transfer with a characteristic transfer time of approximately 5 ps. This finding is in agreement with spectral hole-burning data obtained for bulk samples of Synechocystis PCC 6803. The importance of comparing the results of ensemble (spectral hole burning) and single-complex measurements was demonstrated. The presence of narrow ZPLs near 710 nm in addition to the broad fluorescence band at approximately 730 nm in Thermosynechococcus elongatus (Jelezko et al. J. Phys. Chem. B 2000, 104, 8093-8096) has been confirmed. We also demonstrate that high-quality samples obtained by dissolving crystals of PS I of Thermosynechococcus elongatus exhibit stronger absorption in the red antenna region than any samples studied so far by us and other groups.

Probing Energy Landscapes of Cytochrome b6f with Spectral Hole Burning: Effects of Deuterated Solvent and Detergent.

In non-photochemical spectral hole burning (NPHB) and spectral hole recovery experiments, cytochrome b6f protein exhibits behavior that is almost independent of the deuteration of the buffer/glycerol glassy matrix containing the protein, apart from some differences in heat dissipation. On the other hand, strong dependence of the hole burning properties on sample preparation procedures was observed and attributed to a large increase of the electron-phonon coupling and shortening of the excited-state lifetime occurring when n-dodecyl ß-d-maltoside (DM) is used as a detergent instead of n-octyl ß-d-glucopyranoside (OGP). The data was analyzed assuming that the tunneling parameter distribution or barrier distribution probed by NPHB and encoded into the spectral holes contains contributions from two nonidentical components with accidentally degenerate excited state λ-distributions. Both components likely reflect protein dynamics, although with some small probability one of them (with larger md2) may still represent the dynamics involving specifically the -OH groups of the water/glycerol solvent. Single proton tunneling in the water/glycerol solvent environment or in the protein can be safely excluded as the origin of observed NPHB and hole recovery dynamics. The intensity dependence of the hole growth kinetics in deuterated samples likely reflects differences in heat dissipation between protonated and deuterated samples. These differences are most probably due to the higher interface thermal resistivity between (still protonated) protein and deuterated water/glycerol outside environment.

Frequency-domain spectroscopic study of the PS I-CP43' supercomplex from the cyanobacterium Synechocystis PCC 6803 grown under iron stress conditions.

Absorption, fluorescence excitation, emission, and hole-burning (HB) spectra were measured at liquid helium temperatures for the PS I-CP43' supercomplexes of Synechocystis PCC 6803 grown under iron stress conditions and for respective trimeric PS I cores. Results are compared with those of room temperature, time-domain experiments (Biochemistry 2003, 42, 3893) as well as with the low-temperature steady-state experiments on PS I-CP43' supercomplexes of Synechococcus PCC 7942 (Biochim. Biophys. Acta 2002, 1556, 265). In contrast to the CP43' of Synechococcus PCC 7942, CP43' of Synechocystis PCC 6803 possesses two low-energy states analogous to the quasidegenerate states A and B of CP43 of photosystem II (J. Phys. Chem. B 2000, 104, 11805). Energy transfer between the CP43' and the PS I core occurs, to a significant degree, through the state A, characterized with a broader site distribution function (SDF). It is demonstrated that the low temperature (T = 5 K) excitation energy transfer (EET) time between the state A of CP43' (IsiA) and the PS I core in PS I-CP43' supercomplexes from Synechocystis PCC 6803 is about 60 ps, which is significantly slower than the EET observed at room temperature. Our results are consistent with fast (< or =10 ps) energy transfer from state B to state A in CP43'. Energy absorbed by the CP43' manifold has, on average, a greater chance of being transferred to the reaction center (RC) and utilized for charge separation than energy absorbed by the PS I core antenna. This indicates that energy is likely transferred from the CP43' to the RC along a well-defined path and that the "red antenna states" of the PS I core are localized far away from that path, most likely on the B7-A32 and B37-B38 dimers in the vicinity of the PS I trimerization domain (near PsaL subunit). We argue that the A38-A39 dimer does not contribute to the red antenna region.

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.

Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States.

We explored the rich satellite hole structures emerging as a result of spectral hole burning in cyanobacterial photosystem I (PSI) and demonstrated that hole burning properties of PSI, particularly at high resolution, are strongly affected by the oxidation state of the primary donor P700, as P700+ effectively quenches the excitations of the lowest-energy antenna states responsible for fluorescence. Obtaining better control of this variable will be crucial for high-resolution ensemble experiments on protein energy landscapes in PSI. The separate nonphotochemical spectral hole burning (NPHB) signatures of various red antenna states were obtained, allowing for additional constraints on excitonic structure-based calculations. Preliminary evidence is presented for an additional red state of PSI of T. elongatus peaked at 712.6 nm, distinct from previously reported C708 and C715 states and possibly involving chlorophyll B15. Excitation at wavelengths as long as 800 nm results in charge separation at cryogenic temperatures in PSI also in Synechocystis sp. PCC 6803. Both the "P700+ minus P700" holes and nonphotochemical spectral holes were subjected to thermocycling. The distribution of barriers manifesting in recovery of the "P700+ minus P700" signature contains two components in sample-dependent proportions, likely reflecting the percentages of FA and FB clusters being successfully prereduced before the optical experiment. The barrier distribution for the recovery of the lower-energy nonphotochemical spectral holes resembles those observed for other pigment-protein complexes, suggesting similar structural elements are responsible for NPHB. Higher-energy components exhibit evidence of "domino effects" such as shifts of certain bands persisting past the lower-energy hole recovery. Thus, conformational changes triggered by excitation of one pigment likely can affect multiple pigments in this tightly packed system.

Monte Carlo Modeling of Spectral Diffusion Employing Multiwell Protein Energy Landscapes: Application to Pigment-Protein Complexes Involved in Photosynthesis.

We are reporting development and initial applications of the light-induced and thermally induced spectral diffusion modeling software, covering nonphotochemical spectral hole burning (NPHB), hole recovery, and single-molecule spectroscopy and involving random generation of the multiwell protein energy landscapes. The model includes tunneling and activated barrier-hopping in both ground and excited states of a protein-chromophore system. Evolution of such a system is predicted by solving the system of rate equations. Using the barrier parameters from the range typical for the energy landscapes of the pigment-protein complexes involved in photosynthesis, we (a) show that realistic cooling of the sample must result in proteins quite far from thermodynamic equilibrium, (b) demonstrate hole evolution in the cases of burning, fixed-temperature recovery and thermocycling that mostly agrees with the experiment and modeling based on the NPHB master equation, and (c) explore the effects of different protein energy landscapes on the antihole shape. Introducing the multiwell energy landscapes and starting the hole burning experiments in realistic nonequilibrium conditions are not sufficient to explain all experimental observations even qualitatively. Therefore, for instance, one is required to invoke the modified NPHB mechanism where a complex interplay of several small conformational changes is poising the energy landscape of the pigment-protein system for downhill tunneling.

Conformational Changes in Pigment-Protein Complexes at Low Temperatures-Spectral Memory and a Possibility of Cooperative Effects.

We employed nonphotochemical hole burning (NPHB) and fluorescence line narrowing (FLN) spectroscopies to explore protein energy landscapes and energy transfer processes in dimeric Cytochrome b6f, containing one chlorophyll molecule per protein monomer. The parameters of the energy landscape barrier distributions quantitatively agree with those reported for other pigment-protein complexes involved in photosynthesis. Qualitatively, the distributions of barriers between protein substates involved in the light-induced conformational changes (i.e., -NPHB) are close to glass-like ∼1/√V (V is the barrier height) and not to Gaussian. There is a high degree of correlation between the heights of the barriers in the ground and excited states in individual pigment-protein systems, as well as nearly perfect spectral memory. Both NPHB and hole recovery are due to phonon-assisted tunneling associated with the increase of the energy of a scattered phonon. As the latter is unlikely for simultaneously both the hole burning and the hole recovery, proteins must exhibit a NPHB mechanism involving diffusion of the free volume toward the pigment. Entities involved in the light-induced conformational changes are characterized by md(2) value of about 1.0 × 10(-46) kg·m(2). Thus, these entities are protons or, alternatively, small groups of atoms experiencing sub-Å shifts. However, explaining all spectral hole burning and recovery data simultaneously, employing just one barrier distribution, requires a drastic decrease in the attempt frequency to about 100 MHz. This decrease may occur due to cooperative effects. Evidence is presented for excitation energy transfer between the chlorophyll molecules of the adjacent monomers. The magnitude of the dipole-dipole coupling deduced from the Δ-FLN spectra is in good agreement with the structural data, indicating that the explored protein was intact.