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.

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.

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.

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.

Modeling of various optical spectra in the presence of slow excitation energy transfer in dimers and trimers with weak interpigment coupling: FMO as an example.

We present an improved simulation methodology to describe nonphotochemical hole-burned (NPHB) spectra. The model, which includes both frequency-dependent excitation energy transfer (EET) rate distributions and burning following EET, provides reasonable fits of various optical spectra including resonant and nonresonant holes in the case of FMO complex. A qualitative description of the NPHB process in light of a very complex protein energy landscape is briefly discussed. As an example, we show that both resonant and nonresonant HB spectra obtained for the 825 nm band of the trimeric FMO of C. tepidum are consistent with the presence of a relatively slow EET between the lowest energy states of the monomers of the trimer (mostly localized on BChl a 3), with a weak (∼1 cm(-1)) coupling between these states revealed via calculated emission spectra. We argue that the nature of the so-called 825 nm absorption band of the FMO trimer, contrary to the presently accepted consensus, cannot be explained by a single transition.

On the Controversial Nature of the 825 nm Exciton Band in the FMO Protein Complex.

The nature of the low-energy 825 nm band of the Fenna-Matthews-Olson (FMO) protein complex from Chlorobaculum tepidum at 5 K is discussed. It is shown, using hole-burning (HB) spectroscopy and excitonic calculations, that the 825 nm absorption band of the FMO trimer cannot be explained by a single electronic transition or overlap of electronic transitions of noninteracting pigments. To explain the shape of emission and nonresonant HB spectra, downward uncorrelated excitation energy transfer (EET) between trimer subunits should be taken into account. Modeling studies reveal the presence of three sub-bands within the 825 nm band, in agreement with nonresonant HB and emission spectra. We argue that after light induced coherences vanish, uncorrelated EET between the lowest exciton levels of each monomer takes place. HB induced spectral shifts provide a new insight on the energy landscape of the FMO protein.

Primary electron donor(s) in isolated reaction center of photosystem II from Chlamydomonas reinhardtii.

Isolated reaction centers (RCs) from wild-type Chlamydomonas (C.) reinhardtii of Photosystem II (PSII), at different levels of intactness, were studied to provide more insight into the nature of the charge-separation (CS) pathway(s). We argue that previously studied D1/D2/Cytb559 complexes (referred to as RC680), with ChlD1 serving as the primary electron donor, contain destabilized D1 and D2 polypeptides and, as a result, do not provide a representative model system for the intact RC within the PSII core. The shapes of nonresonant transient hole-burned (HB) spectra obtained for more intact RCs (referred to as RC684) are very similar to P(+)QA(-) - PQA absorbance difference and triplet minus singlet spectra measured in PSII core complexes from Synechocystis PCC 6803 [Schlodder et al. Philos. Trans. R. Soc. London, Ser. B2008, 363, 1197]. We show that in the RC684 complexes, both PD1 and ChlD1 may serve as primary electron donors, leading to two different charge separation pathways. Resonant HB spectra cannot distinguish the CS times corresponding to different paths, but it is likely that the zero-phonon holes (ZPHs) observed in the 680-685 nm region (corresponding to CS times of ∼1.4-4.4 ps) reveal the ChlD1 pathway; conversely, the observation of charge-transfer (CT) state(s) in RC684 (in the 686-695 nm range) and the absence of ZPHs at λB > 685 nm likely stem from the PD1 pathway, for which CS could be faster than 1 ps. This is consistent with the finding of Krausz et al. [Photochem. Photobiol. Sci.2005, 4, 744] that CS in intact PSII core complexes can be initiated at low temperatures with fairly long-wavelength excitation. The lack of a clear shift of HB spectra as a function of excitation wavelength within the red-tail of the absorption (i.e., 686-695 nm) and the absence of ZPHs suggest that the lowest-energy CT state is largely homogeneously broadened. On the other hand, in usually studied destabilized RCs, that is, RC680, for which CT states have never been experimentally observed, ChlD1 is the most likely electron donor.

Spectroscopic study of the CP43' complex and the PSI-CP43' supercomplex of the cyanobacterium Synechocystis PCC 6803.

The PSI-CP43' supercomplex of the cyanobacterium Synechocystis PCC 6803, grown under iron-starvation conditions, consists of a trimeric core Photosystem I (PSI) complex and an outer ring of 18 CP43' light-harvesting complexes. We have investigated the electronic structure and excitation energy transfer (EET) pathways within the CP43' (also known as the isiA gene product) ring using low-temperature absorption, fluorescence, fluorescence excitation, and hole-burning (HB) spectroscopies. Analysis of the absorption spectra of PSI, CP43', and PSI-CP43' complexes suggests that there are 13 chlorophylls (Chls) per CP43' monomer, i.e., a number that was observed in the CP43 complex of Photosystem II (PSII) (Umena, Y. et al. Nature 2011, 473, 55-60). This is in contrast with the recent modeling studies of Zhang et al. (Biochim. Biophys. Acta 2010, 1797, 457-465), which suggested that IsiA likely contains 15 Chls. Modeling studies of various optical spectra of the CP43' ring using the uncorrelated EET model (Zazubovich, V.; Jankowiak, R. J. Lumin. 2007, 127, 245-250) suggest that CP43' monomers (in analogy to the CP43 complexes of the PSII core) also possess two quasi-degenerate low-energy states, A' and B'. The site distribution functions of states A' and B' maxima/full width at half-maximum (fwhm) are at 684 nm/180 cm(-1) and 683 nm/80 cm(-1), respectively. Our analysis shows that pigments mostly contributing to the lowest-energy A' and B' states must be located on the side of the CP43' complex facing the PSI core, a finding that contradicts the model of Zhang et al. but is in agreement with the model suggested by Nield et al. (Biochemistry2003, 42, 3180-3188). We demonstrate that the A'-A' and B'-B' EET between different monomers is possible, though with a slower rate than intramonomer A'-B' and/or B'-A' energy transfer.

On stabilization of a neutral aromatic ligand by π-cation interactions in monoclonal antibodies.

It has been shown that anti-PAH mAb can bind a particular cross-reactant by adopting two distinct "red" and "blue" conformations of its binding sites [N.M. Grubor et al. PNAS 102, 2005, 7453-7458]. In the case of red conformation of pyrene (Py)/anti-PAH mAb (with a broad fluorescence (0,0)-band with fwhm ~140 cm(-1)), the central role in complex formation was played by π-π interactions. The nature of the blue-shifted conformation with very narrow fluorescence (0,0)-band (fwhm ~75 cm(-1)) was left unclear due to the lack of suitable data for comparison. In this work, we suggest spectroscopic and modeling results obtained for the blue conformation of Py in several mAb (including 4D5 mAb) are consistent with π-cation interactions, underscoring the importance of π-cation interaction in ligand binding and stabilization in agreement with earlier modeling studies [J-L. Pellequer, et al. J. Mol. Biol. 302, 2000, 691-699]. We propose considerable narrowing of the fluorescence origin band of ligand in the protein environment could be regarded as a simple indicator of π-cation interactions. Since 4D5 mAb forms only the blue-shifted conformation, while anti-PAH and 8E11 mAbs form both blue- and red-shifted conformations, we suggest mAb interactions, with Py molecules lacking H-bonding functionality, may induce distinct conformations of mAb binding sites that allow binding by π-π and/or π-cation interactions.

Lowest electronic states of the CP47 antenna protein complex of photosystem II: simulation of optical spectra and revised structural assignments.

In this work, we present simulated steady-state absorption, emission, and nonresonant hole burning (HB) spectra for the CP47 antenna complex of photosystem II (PS II) based on fits to recently refined experimental data (Neupane et al. J. Am. Chem. Soc. 2010, 132, 4214). Excitonic simulations are based on the 2.9 Å resolution structure of the PS II core from cyanobacteria (Guskov et al. Nat. Struct. Mol. Biol. 2009, 16, 334), and allow for preliminary assignment of the chlorophylls (Chls) contributing to the lowest excitonic states. The search for realistic site energies was guided by experimental constraints and aided by simple fitting algorithms. The following experimental constraints were used: (i) the oscillator strength of the lowest-energy state should be approximately ≤0.5 Chl equivalents; (ii) the excitonic structure must explain the experimentally observed red-shifted (∼695 nm) emission maximum; and (iii) the excitonic interactions of all states must properly describe the broad (non-line-narrowed, NLN) HB spectrum (including its antihole) whose shape is extremely sensitive to the excitonic structure of the complex, especially the lowest excitonic states. Importantly, our assignments differ significantly from those previously reported by Raszewski and Renger (J. Am. Chem. Soc. 2008, 130, 4431), due primarily to differences in the experimental data simulated. In particular, we find that the lowest state localized on Chl 526 possesses too high of an oscillator strength to fit low-temperature experimental data. Instead, we suggest that Chl 523 most strongly contributes to the lowest excitonic state, with Chl 526 contributing to the second excitonic state. Since the fits of nonresonant holes are more restrictive (in terms of possible site energies) than those of absorption and emission spectra, we suggest that fits of linear optical spectra along with HB spectra provide more realistic site energies.

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.