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A refined definition of the critical micelle concentration is connected to fluorescence methods and applied to test a molecular thermodynamic model of nonionic detergents used for in vitro studies of membrane proteins.

The SecYEG translocon is a channel in bacteria, which provides a passage for secretory proteins across as well as integration of membrane proteins into the plasma membrane. The molecular mechanism, by which SecYEG manages protein transport while preventing water and ion leakage through the membrane, is still controversial. We employed molecular dynamics simulations to assess the contribution of the major structural elements – the plug and the pore ring (PR) – to the sealing of SecYEG in the active state, i.e., with a signal sequence helix occupying the lateral gate. We found, that the PR alone can provide a very tight seal for the wild-type translocon in the active state for both water and ions. Simulations of the mutant I403N, in which one of the PR-defining isoleucine residues is replaced with asparagine, suggest that hydrophobic interactions within the PR and between the PR and the plug are important for maintaining a tight conformation of the wild-type channel around the PR. Disruption of these interactions results in strong fluctuations of helix TM7 and water leakage of the translocon., The hydrophobic interactions between helix TM7, the plug and the pore ring of the translocon determine the barrier to water and ion permission.

The direct usage of the Kramers-Kronig (KK) relations is complicated by two factors: limited frequency range of the available spectra and experimental errors. Here, we reconsider the application of the KK relations to experimental data for the construction of a self-consistent set of optical constants over a wide spectral range: the real part of the complex optical constant, F

Inhomogeneous broadening of optical lines of the Fenna-Matthews-Olson (FMO) light-harvesting protein is investigated by combining a Monte Carlo sampling of low-energy conformational substates of the protein with a quantum chemical/electrostatic calculation of local transition energies (site energies) of the pigments. The good agreement between the optical spectra calculated for the inhomogeneous ensemble and the experimental data demonstrates that electrostatics is the dominant contributor to static disorder in site energies. Rotamers of polar amino acid side chains are found to cause bimodal distribution functions of site energy shifts, which can be probed by hole burning and single-molecule spectroscopy. When summing over the large number of contributions, the resulting distribution functions of the site energies become Gaussians, and the correlations in site energy fluctuations at different sites practically average to zero. These results demonstrate that static disorder in the FMO protein is in the realm of the central limit theorem of statistics.

The photosynthetic apparatus of purple bacteria uses exciton delocalization and static disorder to modulate the position and broadening of its absorption bands, leading to efficient light harvesting. Its main antenna complex, LH2, contains two rings of identical bacteriochlorophyll pigments, B800 and B850, absorbing at 800 and 850 nm, respectively. It has been an unsolved problem why static disorder of the strongly coupled B850 ring is several times larger than that of the B800 ring. Here we show that mixing between excitons and charge transfer states in the B850 ring is responsible for the effect. The linear absorption spectrum of the LH2 system is simulated by using a multiscale approach with an exciton Hamiltonian generalized to include the charge transfer states that involve adjacent pigment pairs, with static disorder modeled microscopically by molecular dynamics simulations. Our results show that sufficient inhomogeneous broadening of the B850 band, needed for efficient light harvesting, is only obtained by utilizing static disorder in the coupling between local excited and interpigment charge transfer states.

Photosystem II (PSII) is a membranespanning, multisubunit pigmentprotein complex responsible for the oxidation of water and the reduction of plastoquinone in oxygenic photosynthesis. In the present review, the recent explosive increase in available structural information about the PSII core complex based on Xray crystallography and cryoelectron microscopy is described at a level of detail that is suitable for a future structurebased analysis of lightharvesting processes. This description includes a proposal for a consistent numbering scheme of proteinbound pigment cofactors across species. The structural survey is complemented by an overview of the state of affairs in structurebased modeling of excitation energy transfer in the PSII core complex with emphasis on electrostatic computations, optical properties of the reaction center, the assignment of longwavelength chlorophylls, and energy trapping mechanisms. (VLID)4919193 Version of record

The origin of the non-conservative nature of the circular dichroism spectrum of the CP29 light-harvesting complex in the Qy spectral region is investigated. A structure-based Hamiltonian of coupled Qy transitions, determined previously [Muh et al., Phys. Chem. Chem. Phys., 2014, 16, 11848] is extended by including higher excited states of the chlorophylls and the S0 → S2 transition of carotenoids. Excitonic couplings are calculated with the Poisson-TrESP method, taking into account dipole strengths from experiments on isolated pigments. The coupling between Qy and higher excited states is found to be responsible for the major part of the non-conservativity of the CD spectrum. The remaining part is explained by the intrinsic CD of the chlorophylls that has been estimated from experiments on isolated pigments.

The file "fmo.pdb" contains the optimized structure of the FMO trimer in pdb format. "BChla.dat" contains the ground and excited state partial charges of the BChla in units of the elementary charge e. Charges were obtained from a fit of the electrostatic potential obtained with (time-dependent) density functional theory on a geometry-optimized bachteriochlorophyll a pigment with the CAM-B3LYP XC-functional and a 6-31G(d,p) basis set. The geometry optimization was performed with DFT and the B3LYP XC functional. "exc_hamiltonian.dat" contains the exction Hamiltonian in units of 1/cm and in the format: H(1,1) H(1,2) ... ... ... H(1,24) H(2,2) H(2,3) ... H(2,24) . . . H(24,24) "frequencies.dat" contains the frequencies of the NMA in units of 1/cm. The first six frequencies (translation and rotation) are omitted. Every line corresponds to one frequency. "g_xi.dat" contains the dimensionless coupling constants g(m)_xi, where m counts the pigments and xi the normal modes, formatted in the following way: g(1)_1 g(2)_1 ... g(24)_1 g(1)_2 g(2)_2 ... g(24)_2 . . . . . . . . . g(1)_59367 g(2)_59367 ... g(24)_59367 The file g_xi.dat has been updated. The old version had a format problem.

We present a microscopic theory for the description of fluctuation-induced excitation energy transfer in chromophore dimers to explain experimental data on a perylene biscarboximide dyad with orthogonal transition dipole moments. Our non-Condon extension of Forster theory takes into account the fluctuations of excitonic couplings linear and quadratic in the normal coordinates, treated microscopically by quantum chemical/electrostatic calculations. The modulation of the optical transition energies of the chromophores is inferred from optical spectra of the isolated chromophores. The application of the theory to the considered dyad reveals a two to three order of magnitude increase in the rate constant by non-Condon effects. These effects are found to be dominated by fluctuations linear in the normal coordinates and provide a structure-based qualitative interpretation of the experimental time constant for energy transfer as well as its dependence on temperature.

We present a structure-based theory of the long-wavelength (red/green) color tuning in visual rhodopsins and its application to the analysis of site-directed mutagenesis experiments. Using a combination of electrostatic and molecular-mechanics methods, we explain the measured mutant-minus-wild-type absorption shifts and conclude that the dominant mechanism of the color tuning in these systems is electrostatic pigment-protein coupling. An important element of our analysis is the independent determination of protonation states of titratable residues in the wild type and the mutant protein as well as the self-consistent reoptimization of hydrogen atom positions, which includes the relaxation of the hydrogen bonding network and the reorientation of water molecules. On the basis of this analysis, we propose a "dipole-orientation rule" according to which both the position and the orientation of a polar group introduced in the protein environment determine the direction of the transition energy shift of the retinal chromophore.

To identify energy traps in CP43, a subcomplex of the photosystem II antenna system, site energies and excitonic couplings of the QY transitions of chlorophyll (Chl) a pigments bound to CP43 are computed using electrostatic models of pigment-protein and pigment-pigment interactions. The computations are based on recent crystal structures of the photosystem II core complex with resolutions of 1.9 and 2.1Å and compared to earlier results obtained at 2.9Å resolution. Linear optical spectra (i.e., absorption, linear dichroism, circular dichroism, and fluorescence) are simulated using the computed excitonic couplings, a refinement fit for the site energies, and a dynamical theory of optical lineshapes. A comparison of the obtained root mean square deviation of about 100 cm(-1) between directly calculated and refined site energies with the maximum range of about 350 cm(-1) of directly calculated site energies shows that the combined quantum chemical/electrostatic approach provides a semi-quantitative agreement with experiment. Possible reasons for the deviations are discussed, including limits of the electrostatic models and the lineshape theory as well as structural alterations of CP43 upon detachment from the core complex. Based on the simulations, an assignment of the two low-energy exciton states A and B of CP43, that where observed earlier in hole burning studies, is suggested. State A is assigned to a localized exciton state on Chl 37 in the lumenal layer of pigments. State B is assigned to an exciton state that is delocalized over several pigments in the cytoplasmic layer. The delocalization explains the smaller inhomogeneous width of state B compared to state A observed in hole burning spectra, which is proposed to be due to exchange narrowing. The assignment of states A and B largely confirms our earlier suggestion that was based on a fit of linear optical spectra and electrostatic calculations using the 2.9Å resolution structure. Interestingly, for the latter structure, the site energy of Chl 37 is obtained closer to the refined value than for 1.9 and 2.1Å resolution. This is explained by a variation of the site energy due to the influence of lipids that might be different in the core complex and isolated CP43. To remove remaining uncertainties in the assignment of states A and B, target sites for mutagenesis experiments are proposed based on the electrostatic computations. In particular, it is suggested to mutate Trp C63 close to Chl 37 to probe the identity of state A and to mutate Arg C41 close to Chl 47 to probe state B.

© 2017 American Chemical Society. Photosystem II (PSII) catalyzes the photo-oxidation of water to molecular oxygen and protons. The water splitting reaction occurs inside the oxygen-evolving complex (OEC) via a Mn4CaO5cluster. To elucidate the reaction mechanism, detailed structural information for each intermediate state of the OEC is required. Despite the current high-resolution crystal structure of PSII at 1.85 Å and other efforts to follow the structural changes of the Mn4CaO5cluster using X-ray free electron laser (XFEL) crystallography in addition to spectroscopic methods, many details about the reaction mechanism and conformational changes in the catalytic site during water oxidation still remain elusive. In this study, we present a rarely found successful application of the conventional macroseeding method to a large membrane protein like the dimeric PSII core complex (dPSIIcc). Combining microseeding with macroseeding crystallization techniques allowed us to reproducibly grow large dPSIIcc crystals with a size of ∼3 mm. These large crystals will help improve the data collected from spectroscopic methods like polarized extended X-ray absorption fine structure (EXAFS) and single crystal electron paramagnetic resonance (EPR) techniques and are a prerequisite for determining a three-dimensional structure using neutron diffraction.

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c(6) and cyt c from horse heart (cyt c(HH)) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt c(HH) than for cyt c(6), the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt c(HH) besides one specific binding site close to P(700). Binding to PS I was found to be the same for reduced and oxidized cyt c(HH). Based on this information, suitable conditions for cocrystallization of cyt c(HH) with PS I were found, resulting in crystals with a PS I:cyt c(HH) ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt c(HH) cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c(6) to PS I revealed a specific binding site where the distance and orientation of cyt c(6) relative to P(700) are comparable with cyt c(2) from purple bacteria relative to P(870). This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I–cyt c costructure and a more detailed understanding of natural electron transport processes.

The fluorescence of monomeric photosystem II core complexes (mPSIIcc) of the cyanobacterium Thermosynechococcus elongatus, originating from redissolved crystals, is investigated by using single-molecule spectroscopy (SMS) at 1.6 K. The emission spectra of individual mPSIIcc are dominated by sharp zero-phonon lines, showing the existence of different emitters compatible with the F685, F689, and F695 bands reported formerly. The intensity of F695 is reduced in single mPSIIcc as compared to single PSIIcc-dimers (dPSIIcc). Crystal structures show that one of the β-carotene (β-Car) cofactors located at the monomer–monomer interface in dPSIIcc is missing in mPSIIcc. This β-Car in dPSIIcc is in van der Waals distance to chlorophyll (Chl) 17 in the CP47 subunit. We suggest that this Chl contributes to the F695 emitter. A loss of β-Car cofactors in mPSIIcc preparations will lead to an increased lifetime of the triplet state of Chl 17, which can explain the reduced singlet emission of F695 as observed in SMS.

SummaryPhotosystem II (PSII) catalyzes a key step in photosynthesis, the oxidation of water to oxygen. Excellent structural models exist for the dimeric PSII core complex of cyanobacteria, but higher order physiological assemblies readily dissociate when solubilized from the native thylakoid membrane with detergent. Here, we describe the crystallization of PSII from Thermosynechococcus elongatus with a postcrystallization treatment involving extraction of the detergent C12E8. This resulted in a transition from Type II to Type I-like membrane protein crystals and improved diffraction to 2.44 Å resolution. The obtained PSII packing in precise rows, interconnected by specific pairs of galactolipids and a loop in the PsbO subunit specific to cyanobacteria, is superimposable with previous electron microscopy images of the thylakoid membrane. The study provides a detailed model of such a superstructure and its organization of light-harvesting pigments with possible implications for the understanding of their efficient use of solar energy.

In order to clarify possibilities to identify the β -carotene ( β -Car) radicals in secondary electron transfer (ET) reactions in the photosystem II core complex (PSIIcc), Raman intensities of all 96 β -Car cofactors in the unit cell of PSIIcc-dimer crystals as a function of polarisation and crystal orientation were simulated based on the 2.9 A resolution structure. The Raman-active symmetry A g in the C 2 h group is assigned to the β -Car modes ν 66 and ν 67 . Simulations are in agreement with experiment for off-resonant excitation at 1064 nm. Resonant measurements at 476 and 532 nm excitation can not be explained, which is attributed to mode mixing in the excited state and the existence of different spectral pools. The identity of the β -Car oxidised in secondary ET can not be resolved by Raman measurements on PSIIcc-dimer crystals. Additional simulations show that similar measurements on PSIIcc-monomer crystals could provide a possible route to solve this issue.

The exciton Hamiltonian of the chlorophyll (Chl) and pheophytin (Pheo) pigments in the reaction center (RC) of photosystem II is computed based on recent crystal structures by using the Poisson–Boltzmann/quantum-chemical method. Computed site energies largely confirm a previous model inferred from fits of optical spectra, in which ChlD1 has the lowest site energy, while that of PheoD1 is higher than that of PheoD2. The latter assignment has been challenged recently under reference to mutagenesis experiments. We argue that these data are not in contradiction to our results. We conclude that ChlD1 is the primary electron donor in both isolated RCs and intact core complexes at least at cryogenic temperatures. The main source of asymmetry in site energies is the charge distribution in the protein. Because many small contributions from various structural elements have to be taken into account, it can be assumed that this asymmetry was established in evolution by global optimization of the RC protein.

© 2016 Elsevier B.V. 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.

Excitation energy transfer in the light-harvesting complex II of higher plants is modeled using excitonic couplings and local transition energies determined from structure-based calculations recently ( Muh et al., 2010 ). A theory is introduced that implicitly takes into account protein induced dynamic localization effects of the exciton wavefunction between weakly coupled optical and vibronic transitions of different pigments. Linear and non-linear optical spectra are calculated and compared with experimental data reaching qualitative agreement. High-frequency intramolecular vibrational degrees of freedom are found important for ultrafast subpicosecond excitation energy transfer between chlorophyll (Chl) b and Chla, since they allow for fast dissipation of the excess energy. The slower ps component of this transfer is due to the monomeric excited state of Chlb 605. The majority of exciton relaxation in the Chla spectral region is characterized by slow ps exciton equilibration between the Chla domains within one layer and between the lumenal and stromal layers in the 10–20 ps time range. Subpicosecond exciton relaxation in the Chla region is only found within the terminal emitter domain (Chls a 610/611/612) and within the Chla 613/614 dimer. Deviations between measured and calculated exciton state life times are obtained for the intermediate spectral region between the main absorbance bands of Chla and Chlb that indicate that besides Chlb 608 another pigment should absorb there. Possible candidates, so far not identified by structure-based calculations, but by fitting of optical spectra and mutagenesis studies, are discussed. Additional mutagenesis studies are suggested to resolve this issue.

Herbicides that target photosystem II (PSII) compete with the native electron acceptor plastoquinone for binding at the Q(B) site in the D1 subunit and thus block the electron transfer from Q(A) to Q(B). Here, we present the first crystal structure of PSII with a bound herbicide at a resolution of 3.2 Å. The crystallized PSII core complexes were isolated from the thermophilic cyanobacterium Thermosynechococcus elongatus. The used herbicide terbutryn is found to bind via at least two hydrogen bonds to the Q(B) site similar to photosynthetic reaction centers in anoxygenic purple bacteria. Herbicide binding to PSII is also discussed regarding the influence on the redox potential of Q(A), which is known to affect photoinhibition. We further identified a second and novel chloride position close to the water-oxidizing complex and in the vicinity of the chloride ion reported earlier (Guskov, A., Kern, J., Gabdulkhakov, A., Broser, M., Zouni, A., and Saenger, W. (2009) Nat. Struct. Mol. Biol. 16, 334-342). This discovery is discussed in the context of proton transfer to the lumen.

The knowledge of the vibrational properties is of key importance to understand their function of β-carotene molecules in the reaction centre of the light-harvesting complexes. We present polarisation-dependent Raman spectroscopy on β-carotene molecules in two different environments: first single dimeric photosystem II crystal, and second encapsulated β-carotene in single-wall carbon nanotube bundles. Both systems have nearly perpendicular or parallel orientated β-carotene molecules. We compare the dependence of their Raman spectra on the polarisation of incident and scattered light.

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C2221, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone QA, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn4Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn4Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.

The photosynthetic oxygen-evolving photosystem II (PSII) is the only known biochemical system that is able to oxidize water molecules and thereby generates almost all oxygen in the Earth's atmosphere. The elucidation of the structural and mechanistic aspects of PSII keeps scientists all over the world engaged since several decades. In this Minireview, we outline the progress in understanding PSII based on the most recent crystal structure at 2.9 A resolution. A likely position of the chloride ion, which is known to be required for the fast turnover of water oxidation, could be determined in native PSII and is compared with work on bromide and iodide substituted PSII. Moreover, eleven new integral lipids could be assigned, emphasizing the importance of lipids for the perfect function of PSII. A third plastoquinone molecule (Q(C)) and a second quinone transfer channel are revealed, making it possible to consider different mechanisms for the exchange of plastoquinone/plastoquinol molecules. In addition, possible transport channels for water, dioxygen and protons are identified.

In photosynthesis the sunlight is collected by antenna pigments and transmitted to special molecule structures called photosystem I (PS I) and photosystem II (PS II), where the energy of light is transformed into a separation of charge. In the PS II reaction centre (RC), there are two β-carotene molecules Car D1 and Car D2 , which are oriented perpendicular to each other. The role of these molecules in the photosynthesis process is still unknown. We use Raman spectroscopy to study the two β-carotenes. We present polarised Raman measurements of the β-carotene vibrational modes in single crystals of photosystem II (PS II) and discuss the possible origin of the observed polarisation dependence. These measurements are important for the basic understanding of the β-carotene vibrational properties and guide future investigations of PS II.

SummaryUsing the 2.9 Å resolution structure of the membrane-intrinsic protein-cofactor complex photosystem II (PSII) from the cyanobacterium Thermosynechococcus elongatus, we calculated and characterized nine possible substrate/product channels leading to/away from the Mn4Ca cluster, where water is oxidized to dioxygen, protons, and electrons. Five narrow channels could function in proton transport, assuming that no large structural changes are associated with water oxidation. Four wider channels could serve to supply water to or remove oxygen from the Mn4Ca cluster. One of them might be regulated by conformational changes of Lys134 in subunit PsbU. Data analyses of Kr derivatized crystals and complexes with dimethyl sulfoxide (DMSO) confirm the accessibility of the proposed dioxygen channels to other molecules. Results from Xe derivatization suggest that the lipid clusters within PSII could serve as a drain for oxygen because of their predominant hydrophobic character and mediate dioxygen release from the lumen.

The excited states of chromophore dimers are, in general, delocalized, and the transition energies and transition dipoles are different from those of the monomers. The intermolecular interaction that is responsible for these effects has two contributions: Forster-type Coulomb coupling and a short-range coupling, which depends on the intermolecular overlap of electronic wave functions. The latter contains the Dexter-type exchange coupling and the coupling of excited states to intermolecular charge-transfer (CT) states. Recently, we developed a method (TrEsp) for an accurate and numerically efficient calculation of the Forster-type Coulomb part (Madjet et al. J. Phys. Chem. B 2006, 110, 17268). Here, we combine the latter with quantum chemical calculations to evaluate the short-range contribution, extending a method developed earlier by Scholes et al. (J. Phys. Chem. B 1999, 103, 2543). An effective two-state model is used, which relates the transition energies and transition dipole moments obtained by quantum chemical calculations of the monomers to those calculated for the dimer. From this relation, the short-range excitonic coupling and effective shifts of the local transition energies due to the coupling to intermolecular CT states can be inferred including a consistency check to evaluate quantum chemical methods that differ in the treatment of electron correlation. The method is applied to the special pairs of the reaction centers of purple bacteria (bRC) and photosystem I (PSI). We find that the short-range coupling represents the dominant contribution to the total excitonic coupling in both special pairs (80% in PSI and 70% in the bRC) and exhibits a monoexponential dependence on the distance between the pi-planes of the pigments with an attenuation factor of 2.8 A(-1). We obtain significant red-shifts of the local transition energies, which show a biexponential distance dependence with one attenuation factor being 2.8 A(-1) and another factor being in the range 0.3-0.7 A(-1) for PSI and 0.8-0.9 A(-1) for bRC. Both effects of the short-range coupling determine the excitation energy sink in the reaction centers at the special pairs.

The crystal structure of the class IIb water-soluble chlorophyll binding protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10 degrees from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostatic calculations suggest that a thermally activated ISC via an excited triplet state T4 is responsible for the latter temperature dependence.

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-beta-D-maltosides (CxG2 with x = 10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC) = n0 [prot], where the constant n0 = (2.0+/-0.3) microM(-1) is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x = 10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.

Photosystem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the water splitting reaction during oxygenic photosynthesis. In the first part of this review, we describe the current state of the crystal structure at 3.0 A resolution of cyanobacterial PSII from Thermosynechococcus elongatus [B. Loll et al., Towards complete cofactor arrangement in the 3.0 A resolution structure of photosystem II, Nature 438 (2005) 1040-1044] with emphasis on the core antenna subunits CP43 and CP47 and the small membrane-intrinsic subunits. The second part describes first the general theory of optical spectra and excitation energy transfer and how the parameters of the theory can be obtained from the structural data. Next, structure-function relationships are discussed that were identified from stationary and time-resolved experiments and simulations of optical spectra and energy transfer processes.

The photosystem II core complex (PSIIcc) is a complicated membrane protein occurring in oxygenic photosynthetic organisms that catalyzes a key reaction, the light-induced oxidation of water. It consists of 17 membrane-spanning α-helical and three membrane-extrinsic protein subunits and binds nearly 100 cofactors. Among them is a heme protein composed of one heme b group ligated by two low molecular weight subunits with one transmembrane helix each, referred to as cytochrome (cyt) b559. Despite extensive research, the role of cyt b559 in PSIIcc is still not known. In the present chapter, the structure of cyt b559 is reviewed based on recent crystallographic work together with its unusually complex redox behavior, and some ideas are discussed concerning a possible function of this unique heme protein.

41 Pags.- 1 Tabl.- 10 Figs. The definitive version is available at: http://www.sciencedirect.com/science/journal/00052728, The identification of low-energy chlorophyll pigments in photosystem II (PSII) is critical to our understanding of the kinetics and mechanism of this important enzyme. We report parallel circular dichroism (CD) and circularly polarized luminescence (CPL) measurements at liquid helium temperatures of the proximal antenna protein CP47. This assembly hosts the lowest-energy chlorophylls in PSII, responsible for the well-known “F695” fluorescence band of thylakoids and PSII core complexes. Our new spectra enable a clear identification of the lowest-energy exciton state of CP47. This state exhibits a small but measurable excitonic delocalization, as predicated by its CD and CPL. Using structure-based simulations incorporating the new spectra, we propose a revised set of site energies for the 16 chlorophylls of CP47. The significant difference from previous analyses is that the lowest-energy pigment is assigned as Chl 612 (alternately numbered Chl 11). The new assignment is readily reconciled with the large number of experimental observations in the literature, while the most common previous assignment for the lowest energy pigment, Chl 627(29), is shown to be inconsistent with CD and CPL results. Chl 612(11) is near the peripheral light-harvesting system in higher plants, in a lumen-exposed region of the thylakoid membrane. The low-energy pigment is also near a recently proposed binding site of the PsbS protein. This result consequently has significant implications for our understanding of the kinetics and regulation of energy transfer in PSII., Financial support from the Australian Research Council (Grants DP110104565 and DP150103137 to E.K.), the Austrian Science Fund (FWF) (Grant P24774–N27 to T.R.) and Spanish MINECO and European FEDER (Grant AGL2014–55300–R to R.P) is gratefully acknowledged.

In photosynthesis, light is captured by antenna proteins. These proteins transfer the excitation energy with almost 100% quantum efficiency to the reaction centers, where charge separation takes place. The time scale and pathways of this transfer are controlled by the protein scaffold, which holds the pigments at optimal geometry and tunes their excitation energies (site energies). The detailed understanding of the tuning of site energies by the protein has been an unsolved problem since the first high-resolution crystal structure of a light-harvesting antenna appeared >30 years ago [Fenna RE, Matthews BW (1975) Nature 258:573–577]. Here, we present a combined quantum chemical/electrostatic approach to compute site energies that considers the whole protein in atomic detail and provides the missing link between crystallography and spectroscopy. The calculation of site energies of the Fenna–Matthews–Olson protein results in optical spectra that are in quantitative agreement with experiment and reveals an unexpectedly strong influence of the backbone of two α-helices. The electric field from the latter defines the direction of excitation energy flow in the Fenna–Matthews–Olson protein, whereas the effects of amino acid side chains, hitherto thought to be crucial, largely compensate each other. This result challenges the current view of how energy flow is regulated in pigment–protein complexes and demonstrates that attention has to be paid to the backbone architecture.

The Fenna-Matthews-Olson (FMO) protein of green sulfur bacteria represents an important model protein for the study of elementary pigment-protein couplings. We have previously used a simple approach [Adolphs and Renger (2006) Biophys J 91:2778-2797] to study the shift in local transition energies (site energies) of the FMO protein of Prosthecochloris aestuarii by charged amino acid residues, assuming a standard protonation pattern of the titratable groups. Recently, we have found strong evidence that besides the charged amino acids also the neutral charge density of the protein is important, by applying a combined quantum chemical/electrostatic approach [Müh et al. (2007) Proc Natl Acad Sci USA, in press]. Here, we extract the essential parts from this sophisticated method to obtain a relatively simple method again. It is shown that the main contribution to the site energy shifts is due to charge density coupling (CDC) between the pigments and their pigment, protein and water surroundings and that polarization effects for qualitative considerations can be approximated by screening the Coulomb coupling by an effective dielectric constant.

The Fenna−Matthews−Olson (FMO) light-harvesting protein connects the outer antenna system (chlorosome/baseplate) with the reaction center complex in green sulfur bacteria. Since its first structure determination in the mid-70s, this pigment−protein complex has become an important model system to study excitation energy transfer. Recently, an additional bacteriochlorophyll a (the eighth) pigment was discovered in each subunit of this homotrimer. Our structure-based calculations of the optical properties of the FMO protein demonstrate that the eighth pigment is the linker to the baseplate, confirming recent suggestions from crystallographic studies.

The frozen solution electron paramagnetic resonance spectrum of the primary donor cation P + in reaction centers of site-directed mutants of Rhodobacter ( Rb. ) sphaeroides has been obtained at a microwave frequency ν =360 GHz and a magnetic field B 0 =12.8 T. Due to the high Zeeman resolution of the powder pattern, all three principal components of the rhombic g -tensors at T =160 K could be determined with high accuracy. We compare spectra of the site-directed mutants, in which the axial ligand histidine M202 of the primary donor is replaced by glutamic acid (HE(M202)) or leucine (HL(M202)), with those of the strain R26, whose primary donor is similar to that of the wild type and only lacks the carotenoid. For HE(M202), this is the first determination of its g -tensor with the principal components g xx =2.00335(3), g yy =2.00236(2) and g zz =2.00191(2). While in R26 the primary donor is a bacteriochlorophyll a dimer, the HL(M202) and HE(M202) mutants have previously been shown to be bacteriochlorophyll:bacteriopheophytin heterodimers. Their g -tensor anisotropy Δ g = g xx − g zz shows significant variations in opposite directions when compared with R26, with an increased anisotropy for HE(M202) and a decreased one for HL(M202). Calculations employing Density Functional Theory suggest that the observed shifts originate in different torsional angles of the acetyl group attached to the spin-carrying bacteriochlorophyll half L of the dimer.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. With the aim of better understanding the phase behavior of alkyl maltosides (n-alkyl-beta-D-maltosides, C(n)G(2)) under the conditions of membrane protein crystallization, we studied the influence of poly(ethylene glycol) (PEG) 2000, a commonly used precipitating agent, on the critical micelle concentration (CMC) of the alkyl maltosides by systematic variation of the number n of carbon atoms in the alkyl chain (n = 10, 11, and 12) and the concentration of PEG2000 (chi) in a buffer suitable for the crystallization of cyanobacterial photosystem II. CMC measurements were based on established fluorescence techniques using pyrene and 8-anilinonaphthalene-1-sulfonate (ANS). We found an increase of the CMC with increasing PEG concentration according to ln(CMC/CMC0) = k(P)chi, where CMC0 is the CMC in the absence of PEG and k(P) is a constant that we termed the "polymer constant". In parallel, we measured the influence of PEG2000 on the surface tension of detergent-free buffer solutions. At PEG concentrations chi > 1% w/v, the surface pressure pi(s)(chi) = gamma(0) - gamma(chi) was found to depend linearly on the PEG concentration according to pi(s)(chi) = kappa chi + pi(s)(0), where gamma(0) is the surface tension in the absence of PEG. Based on a molecular thermodynamic modeling, CMC shifts and surface pressure due to PEG are related, and it is shown that k(P) = kappa c(n) + eta, where c(n) is a detergent-specific constant depending inter alia on the alkyl chain length n and eta is a correction for molarity. Thus, knowledge of the surface pressure in the absence of a detergent allows for the prediction of the CMC shift. The PEG effect on the CMC is discussed concerning its molecular origin and its implications for membrane protein solubilization and crystallization.

The photosynthetic reaction center (RC) is an integral membrane protein that carries out the initial charge-separation reactions of photosynthesis. Upon light excitation, a pair (P) of bacteriochlorophylls (Bchls) donates an electron to a bacteriopheophytin (HL), generating an ion-pair state (P+HL-). Previous ENDOR studies of RCs from the purple bacterium Rhodobacter sphaeroides have shown that the unpaired electron of P+ is distributed unequally between the two Bchls of P, with about 2/3 of the unpaired spin and positive charge residing on the Bchl bound to subunit L (PL) and 1/3 on the Bchl bound to M (PM). To investigate the protein's role in establishing the energies of the cations PL+ and PM+ through long-range electrostatic interactions, we mutated Arg L135 and Arg M164 individually to Leu or Glu and measured the effects on the Special TRIPLE and FTIR spectra of P+. These highly conserved residues occupy homologous positions on either side of P but have no hydrogen bonds or steric interactions with ...

To study the specific influence of the protein environment in bacterial photosynthetic reaction centers on the oxidation potential and the spin density distribution of the primary electron donor (P...

The photochemically trapped bacteriopheophytin (BPh) b radical anion in the active branch (Φ A − ) of reaction centers (RCs) from Blastochloris (formerly called Rhodopseudomonas ) viridis is characterized by 1 H-ENDOR as well as optical absorption spectroscopy. The two site-directed mutants YF(M208) and YL(M208), in which tyrosine at position M208 is replaced by phenylalanine and leucine, respectively, are investigated and compared with the wild type. The residue at M208 is in close proximity to the primary electron donor, P, the monomeric bacteriochlorophyll (BChl), B A , and the BPh, Φ A , that are involved in the transmembrane electron transfer to the quinone, Q A , in the RC. The analysis of the ENDOR spectra of Φ A − at 160 K indicates that two distinct states of Φ A − are present in the wild type and the mutant YF(M208). Based on a comparison with Φ A − in RCs of Rhodobacter sphaeroides the two states are interpreted as torsional isomers of the 3-acetyl group of Φ A . Only one Φ A − state occurs in the mutant YL(M208). This effect of the leucine residue at position M208 is explained by steric hindrance that locks the acetyl group in one specific position. On the basis of these results, an interpretation of the optical absorption difference spectrum of the state Φ A − Q A − is attempted. This state can be accumulated at 100 K and undergoes an irreversible change between 100 and 200 K [Tiede et al., Biochim. Biophys. Acta 892 (1987) 294–302]. The corresponding absorbance changes in the BChl Q x and Q y regions observed in the wild type also occur in the YF(M208) mutant but not in YL(M208). The observed changes in the wild type and YF(M208) are assigned to RCs in which the 3-acetyl group of Φ A changes its orientation. It is concluded that this distinct structural relaxation of Φ A can significantly affect the optical properties of B A and contribute to the light-induced absorption difference spectra.

Femtosecond time resolved absorbance changes in the near infrared spectral region were recorded in photosynthetic reaction centers (RCs) of Rhodobacter ( Rb .) sphaeroides and Chloroflexus aurantiacus . For Rb. sphaeroides , wild type and site directed mutant RCs, where the tyrosine at the M210 position was replaced by phenylalanine and leucine, were investigated. In the mutant RCs the decay of the stimulated emission signal was drastically slower and displayed strong multiexponentiality. In the spectral range between 1000 and 1100 nm, the existence of a fast kinetic component with a time constant of 1 ps could be demonstrated. Based on the spectral and dichroic characteristics of the data, we suggest a weakly populated P + B A - state as first electron transfer intermediate in these mutants. The results indicate that primary electron transfer follows a stepwise mechanism, even in the case of an energetically increased primary acceptor. Investigations of the electronic structure of the photooxidized primary donor using ENDOR/TRIPLE spectroscopy demonstrated that changes at the position M210 mainly affect B A and not P.

The effects of detergents on the electronic structure of the oxidized primary donor P+ and the time constant τAP of the P+Q A − charge recombination at ambient temperatures have been investigated in native and mutant reaction centers (RCs) from Rhodobacter sphaeroides. It is shown that N-lauryl-N,N-dimethyl-3-ammonio-1-propane sulfonate (SB12) induces a transition to a second distinct conformation of the RC. In the case of the wild type and the mutant FY(M197), in which a hydrogen bond is introduced to the 2-acetyl group of the dimer half of P that is associated with the M-subunit of the RC, the conformational change causes a more asymmetric spin density distribution between the two bacteriochlorophyll moieties of P+ in favor of the L-half. For both types of RCs the time constant τAP depends on the SB12/RC ratio as does the position of the long-wavelength band of P, λmax. The increase of τAP by ∼30 ms and the shift of λmax from ∼866 nm to ∼851 nm are indicative for the conformational change. In addition, a smaller linear increase of τAP with increasing SB12/RC ratio is superimposed on the variation of τAP due to the conformational change. Similar effects of SB12 on the optical spectra as well as on τAP are also observed for the two heterodimer mutants HL(L173) and HL(M202), in which one of the bacteriochlorophylls of P is replaced by a bacteriopheophytin. There are no clear indications for a correlation of τAP with the localization of the positive charge in P+. Furthermore, it is concluded from the dependence of τAP on the SB12/RC ratio that the single-site mutations do not affect the standard free energy difference of the two conformations to a measurable extent.

The exciton Hamiltonian pertaining to the first excited states of chlorophyll (Chl) a and b pigments in the minor light-harvesting complex CP29 of plant photosystem II is determined based on the recent crystal structure at 2.8 Å resolution applying a combined quantum chemical/electrostatic approach as used earlier for the major light-harvesting complex LHCII. Two electrostatic methods for the calculation of the local transition energies (site energies), referred to as the Poisson-Boltzmann/quantum chemical (PBQC) and charge density coupling (CDC) method, which differ in the way the polarizable environment of the pigments is described, are compared and found to yield comparable results, when tested against fits of measured optical spectra (linear absorption, linear dichroism, circular dichroism, and fluorescence). The crystal structure shows a Chl a/b ratio of 2.25, whereas a ratio between 2.25 and 3.0 can be estimated from the simulation of experimental spectra. Thus, it is possible that up to one Chl b is lost in CP29 samples. The lowest site energy is found to be located at Chl a604 close to neoxanthin. This assignment is confirmed by the simulation of wild-type-minus-mutant difference spectra of reconstituted CP29, where a tyrosine residue next to Chl a604 is modified in the mutant. Nonetheless, the terminal emitter domain (TED), i.e. the pigments contributing mostly to the lowest exciton state, is found at the Chl a611-a612-a615 trimer due to strong excitonic coupling between these pigments, with the largest contributions from Chls a611 and a612. A major difference between CP29 and LHCII is that Chl a610 is not the energy sink in CP29, which is presumably to a large extent due to the replacement of a lysine residue with alanine close to the TED.

In photosynthesis, specialized pigment–protein assemblies, termed light-harvesting complexes (LHCs) or antenna proteins, absorb solar photons and deliver the excitation energy by exciton transfer (XT) to the photochemically active centers. To understand this process, it has to be linked to molecular structures and spectroscopic properties of LHCs. In this chapter, we show how this link is provided by theoretical modeling. We first describe what excitons are and how the mechanism of XT is influenced by the coupling of excited states to molecular vibrations. This description defines key parameters that are important for a modeling of XT: (1) site energies, (2) excitonic couplings, and (3) exciton-vibrational coupling constants. Next, we discuss how these parameters can be calculated from a crystal structure of the LHC within the framework of an electrostatic model of intermolecular interactions. Finally, we show applications to the Fenna–Matthews–Olson (FMO) protein of green sulfur bacteria and the major LHC of higher plants (LHCII) to illustrate how this approach works and what has already been learned from it.

The effects of zwitterionic detergents on the spectroscopic properties of the primary electron donor P of photosynthetic reaction centers (RCs) from the purple nonsulfur bacterium Rhodobacter sphaeroides are investigated. When RCs are solubilized with N-(n-alkyl)-N,N-dimethyl-3-ammonio-1-propanesulfonates (sulfobetaines) the maximum of the characteristic near-infrared absorption of P, λ max , is shifted to lower wavelengths at room temperature as compared with nonionic detergents. While the range of the blueshift (λ max = 850- 862 nm) is identical for different sulfobetaines, the dependence of the shift on the detergent/RC ratio varies systematically with the number of carbon atoms in the n-alkyl-chain. N-lauryl-N,N-dimethyl-amine-N-oxide (LDAO), a zwitterionic detergent with a less polar head group, is only able to induce blueshifts of λ max down to 859 nm at much higher detergent/RC ratios. ENDOR experiments performed on the primary donor radical cation P + reveal that two distinct states of P exist in such samples. Thus the optical bandshifts are caused by transitions between two spectroscopic species, called P 866 and P 850 . We interpret the detergent effect as an electrostatic influence of the detergent head groups on the equilibrium between two protein conformations which in turn affect the structure of the primary donor.