Effects of distribution of excitation energy transfer times and protein dynamics on spectral hole burning in pigment-protein complexes involved in photosynthesis

Understanding the spectral properties of natural photosynthetic complexes (the excited state lifetimes, electron-phonon couplings, distributions of “solvent shifts” of various pigments, and the interactions between them (manifestations of excitonic effects, excitation energy transfer, charge transfer) as well as pigments site energies, lowest-energy states and origin of various emission bands) is fundamental to advance the design of the artificial photosynthetic systems. Traditionally the spectral properties of natural photosynthetic complexes are explored by either time-domain or frequency-domain high-resolution spectroscopy methods, including non-photochemical spectral hole burning (NPHB). The main goal of this thesis was the study of various effects of the distribution of excitation energy transfer times and protein dynamics on non-photochemical hole burning processes in photosynthetic pigment-protein complexes. In the first part of this thesis we present our results concerning the inclusion of the distributions of excitation energy transfer (EET) rates (homogeneous line widths) and charge separation rates into treatment of the resonant and non-resonant NPHB processes in photosynthetic chlorophyll-protein complexes. Thus, the effects of the line width distributions resulting from Förster-type EET between weakly interacting pigments with uncorrelated site distribution functions, on the resonant NPHB process have been explored both theoretically and experimentally in isolated CP43 antenna from spinach. Furthermore, we have also demonstrated that inclusion of the effects of frequency-dependent EET rate distributions and burning following EET on the treatment of non-resonant NPHB spectra of trimeric Fenna-Matthews-Olson protein from Chlorobium tepidum leads to reasonable agreement between the theoretical and experimental data. The second part of this thesis is focused on the analysis of HB spectral properties of the lowest energy states of Photosystem I (PSI) with the aims to gain better understanding of particular structural origins of these states as well as on the protein dynamics of PSI. We explored the satellite hole structures obtained after illumination at various wavelengths and the dependence of those structures on thermocycling. In order to explore the protein dynamics in PSI SHB experiments and compare it with SPCS observations special attention was devoted to the study of the influence of the P700 redox state on resonant and nonresonant NPHB spectra from cyanobacteria Thermosynechococcus elongatus

Optical spectroscopy of photosynthetic complexes : focus on low-temperature protein dynamics

To perform photosynthesis, plants, algae and bacteria possess well organized and closely coupled photosynthetic pigment-protein complexes. The information on energy transfer processes and protein dynamics contained in the narrow zero-phonon lines at low temperatures is hidden under the inhomogeneous broadening. Thus, it is difficult to analyze the spectroscopic properties of these complexes in sufficient detail by conventional spectroscopy methods. In this context, high resolution spectroscopy techniques such as Spectral Hole Burning, Fluorescence Line Narrowing and Single Molecule / Single Complex Spectroscopy are powerful tools designed to overcome the inhomogeneous broadening difficulty. This thesis focuses mainly on the low-temperature protein dynamics of several photosynthetic protein complexes (LH2, CP43, CP29 and LHCII). The hole growth kinetics and the shape of the anti-hole due to the non-photochemical spectral hole burning have been explored, and interpreted within the framework of theoretical models describing spectral diffusion due to conformational changes between nearly identical substates on a multi-tier protein energy landscapes

Parameters of the Protein Energy Landscapes of Several Light-Harvesting Complexes Probed via Spectral Hole Growth Kinetics Measurements

44 Pag., 2 Tabl. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkThe parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (1 cm−1) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results.Research at Concordia University is supported by NSERC and CFI. R.P. would like to thank Spanish MICINN (grant AGL2008-00377). M.S. acknowledges the contribution of the Photosynthetic Systems Program, Chemical Sciences, Geosciences, and Biosciences Division, Basic Energy Sciences, USDOE. J.P. and K.-D.I. gratefully acknowledge support from Deutsche Forschungsgemeinschaft (SFB 429, TP A1, and TP A3, respectively).Peer reviewe

Conformational changes in pigment-protein complexes at low temperatures - spectral memory and a possibility of cooperative effects

40 Pags.- 9 Figs. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkWe employed non-photochemical 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 sub-states 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 hole recovery, proteins must exhibit a NPHB mechanism involving diffusion of the free volume towards the pigment. Entities involved in the light-induced conformational changes are characterized by md2 value of about 1.0.10-46 kg.m2. 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 delta-FLN spectra is in good agreement with the structural data, indicating that explored protein was intact.Concordia researchers express gratitude to NSERC, CFI and Concordia University. We would also like to acknowledge the financial support by the MINECO (Grant AGL2011-23574) to R. P.Peer reviewe

Parameters of the Protein Energy Landscapes of Several Light-Harvesting Complexes Probed via Spectral Hole Growth Kinetics Measurements

44 Pag., 2 Tabl. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkThe parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (1 cm−1) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results.Research at Concordia University is supported by NSERC and CFI. R.P. would like to thank Spanish MICINN (grant AGL2008-00377). M.S. acknowledges the contribution of the Photosynthetic Systems Program, Chemical Sciences, Geosciences, and Biosciences Division, Basic Energy Sciences, USDOE. J.P. and K.-D.I. gratefully acknowledge support from Deutsche Forschungsgemeinschaft (SFB 429, TP A1, and TP A3, respectively).Peer reviewe

Effects of the distributions of energy or charge transfer rates on spectral hole burning in pigment-protein complexes at low temperatures

51 Pags, 2 Tabls., 7 Figs. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkEffects of the distributions of excitation energy transfer (EET) rates (homogeneous line widths) on the non-photochemical (resonant) spectral hole burning (SHB) processes in photosynthetic chlorophyll-protein complexes (reaction center [RC] and CP43 antenna of Photosystem II from spinach) are considered. It is demonstrated that inclusion of such a distribution results in somewhat more dispersive hole burning kinetics. More importantly, however, inclusion of the EET rate distributions strongly affects the dependence of the hole width on the fractional hole depth. Different types of line width distributions have been explored, including those resulting from Förster type EET between weakly interacting pigments as well as Gaussian ones, which may be a reasonable approximation for those resulting, for instance, from so-called extended Förster models. For Gaussian line width distributions it is possible to determine the parameters of both line width and tunneling parameter distributions from SHB data without a priori knowledge of any of them. Concerning more realistic asymmetric distributions, we demonstrate, using the simple example of CP43 antenna, that one can use SHB modeling to estimate electrostatic couplings between pigments and support or exclude assignment of certain pigment(s) to a particular state.Financial support from NSERC, CFI, Concordia University and Libyan government (S.A.) is gratefully acknowledged. R.P. thanks the MICINN (Grant AGL2008-00377) and the EU FEDER Program (AGL2008-00377) in Spain, and M.S. the US Department of Energy’s Photosynthetic Systems Program within the Chemical Sciences, Geoscience, and Biosciences Division of the Office of Basic Energy Sciences under NREL Contract #DEAC36- 08-GO28308 for support. R.J. acknowledges support from the NSF under grant CHE-0907958.Peer reviewe

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 <i>C. tepidum</i> 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 <i>a</i> 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

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 <i>T. elongatus</i> 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 <i>Synechocystis</i> 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 F_A and F_B 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

Spectral hole burning, recovery, and thermocycling in chlorophyll-protein complexes: Distributions of barriers on the protein energy landscape

40 Pags. The definitive version, with tabls. and figs., is available at: http://pubs.acs.org/journal/jpcbfkChlorophyll–protein complexes are ideal model systems for protein energy landscape research. Here pigments, used in optical spectroscopy experiments as sensitive probes to local dynamics, are built into protein by Nature (in a large variety of local environments; without extraneous chemical manipulations or genetic engineering). Distributions of the tunneling parameter, λ, and/or protein energy landscape barrier heights, V, have been determined for (the lowest energy state of) the CP43 core antenna complex of photosystem II. We demonstrate that spectral hole burning (SHB) and hole recovery (HR) measurements are capable of delivering important information on protein energy landscape properties and spectral diffusion mechanism details. In particular, we show that tunneling rather than barrier hopping is responsible for both persistent SHB and subsequent HR at 5–12 K, which allows us to estimate the md2 parameter of the tunneling entities as 1.0 × 10–46 kg·m2. The subdistributions of λ actually contributing to the nonsaturated spectral holes (and affecting their recovery) differ from the respective full true distributions. In the case of the full λ-distribution being uniform (or the barrier height distribution 1/√V, a model which has been widely employed in theories of amorphous solids at low temperatures and in HR analysis), the difference is qualitative, with λ subdistributions probed in the HR experiments being highly asymmetrical, and barrier V subdistributions deviating significantly from 1/√V. Thus, the distribution of λ for the protein energy landscape tier directly probed by SHB is likely Gaussian and not uniform. Additionally, a Gaussian distribution of barriers, with parameters incompatible with those of the landscape tier directly probed by SHB, contributes to the thermocycling results.Financial support from NSERC, CFI, and Concordia University is gratefully acknowledged. R.P. thanks the MINECO of Spain (Grant AGL2011-23574, partially financed by the EU FEDER Program), and M.S. acknowledges the U.S. Department of Energy’s Photosynthetic Systems Program within the Chemical Sciences, Geoscience, and Biosciences Division of the Office of Basic Energy Sciences under NREL Contract #DEAC36- 08-GO28308 for support. R.J. acknowledges support from the NSF ARRA Grant (CHE-0907958). M.S. also acknowledges partial support from NREL pension program.Peer reviewe