Towards understanding the crystallization of photosystem II: influence of poly(ethylene glycol) of various molecular sizes on the micelle formation of alkyl maltosides.

The influence of poly(ethylene glycol) (PEG) polymers H-(O-CH2-CH2)p-OH with different average molecular sizes p on the micelle formation of n-alkyl-ß-D-maltoside detergents with the number of carbon atoms in the alkyl chain ranging from 10 to 12 is investigated with the aim to learn more about the detergent behavior under conditions suitable for the crystallization of the photosynthetic pigment-protein complex photosystem II. PEG is shown to increase the critical micelle concentration (CMC) of all three detergents in the crystallization buffer in a way that the free energy of micelle formation increases linearly with the concentration of oxyethylene units (O-CH2-CH2) irrespective of the actual molecular weight of the polymer. The CMC shift is modeled by assuming for simplicity that it is dominated by the interaction between PEG and detergent monomers and is interpreted in terms of an increase of the transfer free energy of a methylene group of the alkyl chain by 0.2 kJ mol-1 per 1 mol L-1 increase of the concentration of oxyethylene units at 298 K. Implications of this effect for the solubilization and crystallization of protein-detergent complexes as well as detergent extraction from crystals are discussed.

Living on the edge: light-harvesting efficiency and photoprotection in the core of green sulfur bacteria.

Photosynthetic green sulfur bacteria are able to survive under extreme low light conditions. Nevertheless, the light-harvesting efficiencies reported so far, in particular for Fenna-Matthews-Olson (FMO) protein-reaction center complex (RCC) supercomplexes, are much lower than for photosystems of other species. Here, we approach this problem with a structure-based theory. Compelling evidence for a light-harvesting efficiency around 95% is presented for native (anaerobic) conditions that can drop down to 47% when the FMO protein is switched into a photoprotective mode in the presence of molecular oxygen. Light-harvesting bottlenecks are found between the FMO protein and the RCC, and the antenna of the RCC and its reaction center (RC) with forward energy transfer time constants of 39 ps and 23 ps, respectively. The latter time constant removes an ambiguity in the interpretation of time-resolved spectra of RCC probing primary charge transfer and provides strong evidence for a transfer-to-the trap limited kinetics of excited states. Different factors influencing the light-harvesting efficiency are investigated. A fast primary electron transfer in the RC is found to be more important for a high efficiency than the site energy funnel in the FMO protein, quantum effects of nuclear motion, or variations in the mutual orientation between the FMO protein and the RCC.

Structural determinants of a permeation barrier of the SecYEG translocon in the active state.

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.

Normal mode analysis of spectral density of FMO trimers: Intra- and intermonomer energy transfer.

The intermolecular contribution to the spectral density of the exciton-vibrational coupling of the homotrimeric Fenna-Matthews-Olson (FMO) light-harvesting protein of green sulfur bacteria P. aestuarii is analyzed by combining a normal mode analysis of the protein with the charge density coupling method for the calculation of local transition energies of the pigments. Correlations in site energy fluctuations across the whole FMO trimer are found at low vibrational frequencies. Including, additionally, the high-frequency intrapigment part of the spectral density, extracted from line-narrowing spectra, we study intra- and intermonomer exciton transfer. Whereas the intrapigment part of the spectral density is important for fast intramonomer exciton relaxation, the intermolecular contributions (due to pigment-environment coupling) determine the intermonomer exciton transfer. Neither the variations of the local Huang-Rhys factors nor the correlations in site energy fluctuations have a critical influence on energy transfer. At room temperature, the intermonomer transfer in the FMO protein occurs on a 10 ps time scale, whereas intramonomer exciton equilibration is roughly two orders of magnitude faster. At cryogenic temperatures, intermonomer transfer limits the lifetimes of the lowest exciton band. The lifetimes are found to increase between 20 ps in the center of this band up to 100 ps toward lower energies, which is in very good agreement with the estimates from hole burning data. Interestingly, exciton delocalization in the FMO monomers is found to slow down intermonomer energy transfer, at both physiological and cryogenic temperatures.

Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus.

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) 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 cHH than for cyt c6, 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 cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH 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 c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 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 lowest-energy chlorophyll of photosystem II is adjacent to the peripheral antenna: Emitting states of CP47 assigned via circularly polarized luminescence.

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.

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

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

Origin of non-conservative circular dichroism of the CP29 antenna complex of photosystem II.

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 [Müh 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.

Role of missing carotenoid in reducing the fluorescence of single monomeric photosystem II core complexes.

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 ß-carotene (ß-Car) cofactors located at the monomer-monomer interface in dPSIIcc is missing in mPSIIcc. This ß-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 ß-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.

The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides used in membrane protein crystallization.

With the aim of better understanding the phase behavior of alkyl maltosides (n-alkyl-ß-d-maltosides, CnG2) 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 (χ) 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) = kPχ, where CMC0 is the CMC in the absence of PEG and kP 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 χ > 1% w/v, the surface pressure πs(χ) = γ(0) - γ(χ) was found to depend linearly on the PEG concentration according to πs(χ) = κχ + πs(0), where γ(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 kP = κc(n) + η, where c(n) is a detergent-specific constant depending inter alia on the alkyl chain length n and η 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.

Towards a structure-based exciton Hamiltonian for the CP29 antenna of photosystem II.

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.

Refined structure-based simulation of plant light-harvesting complex II: linear optical spectra of trimers and aggregates.

Linear optical spectra of solubilized trimers and small lamellar aggregates of the major light-harvesting complex II (LHCII) of higher plants are simulated employing excitonic couplings and site energies of chlorophylls (Chls) computed on the basis of the two crystal structures by a combined quantum chemical/electrostatic approach. A good agreement between simulation and experiment is achieved (except for the circular dichroism in the Chl b region), if vibronic transitions of Chls are taken into account. Site energies are further optimized by refinement fits of optical spectra. The differences between refined and directly calculated values are not significant enough to decide, whether the crystal structures are closer to trimers or aggregates. Changes in the linear dichroism spectrum upon aggregation are related to site energy shifts of Chls b601, b607, a603, a610, and a613, and are interpreted in terms of conformational changes of violaxanthin and the two luteins involving their ionone rings. Chl a610 is the energy sink at 77K in both conformations. An analysis of absorption spectra of trimers perpendicular and parallel to the C(3)-axis (van Amerongen et al. Biophys. J. 67 (1994) 837-847) shows that only Chl a604 close to neoxanthin is significantly reoriented in trimers compared to the crystal structures. Whether this pigment is orientated in aggregates as in the crystal structures, can presently not be determined faithfully. To finally decide about pigment reorientations that could be of relevance for non-photochemical quenching, further polarized absorption and fluorescence measurements of aggregates or detergent-depleted LHCII would be helpful. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Light-induced quinone reduction in photosystem II.

The photosystem II core complex is the water:plastoquinone oxidoreductase of oxygenic photosynthesis situated in the thylakoid membrane of cyanobacteria, algae and plants. It catalyzes the light-induced transfer of electrons from water to plastoquinone accompanied by the net transport of protons from the cytoplasm (stroma) to the lumen, the production of molecular oxygen and the release of plastoquinol into the membrane phase. In this review, we outline our present knowledge about the "acceptor side" of the photosystem II core complex covering the reaction center with focus on the primary (Q(A)) and secondary (Q(B)) quinones situated around the non-heme iron with bound (bi)carbonate and a comparison with the reaction center of purple bacteria. Related topics addressed are quinone diffusion channels for plastoquinone/plastoquinol exchange, the newly discovered third quinone Q(C), the relevance of lipids, the interactions of quinones with the still enigmatic cytochrome b559 and the role of Q(A) in photoinhibition and photoprotection mechanisms. This article is part of a Special Issue entitled: Photosystem II.

The nonheme iron in photosystem II.

Photosystem II (PSII), the light-driven water:plastoquinone (PQ) oxidoreductase of oxygenic photosynthesis, contains a nonheme iron (NHI) at its electron acceptor side. The NHI is situated between the two PQs QA and QB that serve as one-electron transmitter and substrate of the reductase part of PSII, respectively. Among the ligands of the NHI is a (bi)carbonate originating from CO2, the substrate of the dark reactions of oxygenic photosynthesis. Based on recent advances in the crystallography of PSII, we review the structure of the NHI in PSII and discuss ideas concerning its function and the role of bicarbonate along with a comparison to the reaction center of purple bacteria and other enzymes containing a mononuclear NHI site.

Structure-based modeling of energy transfer in photosynthesis.

We provide a minimal model for a structure-based simulation of excitation energy transfer in pigment-protein complexes (PPCs). In our treatment, the PPC is assembled from its building blocks. The latter are defined such that electron exchange occurs only within, but not between these units. The variational principle is applied to investigate how the Coulomb interaction between building blocks changes the character of the electronic states of the PPC. In this way, the standard exciton Hamiltonian is obtained from first principles and a hierarchy of calculation schemes for the parameters of this Hamiltonian arises. Possible extensions of this approach are discussed concerning (i) the inclusion of dispersive site energy shifts and (ii) the inclusion of electron exchange between pigments. First results on electron exchange within the special pair of photosystem II of cyanobacteria and higher plants are presented and compared with earlier results on purple bacteria. In the last part of this mini-review, the coupling of electronic and nuclear degrees of freedom is considered. First, the standard exciton-vibrational Hamiltonian is parameterized with the help of a normal mode analysis of the PPC. Second, dynamical theories are discussed that exploit this Hamiltonian in the study of dissipative exciton motion.

Understanding photosynthetic light-harvesting: a bottom up theoretical approach.

We discuss a bottom up approach for modeling photosynthetic light-harvesting. Methods are reviewed for a full structure-based parameterization of the Hamiltonian of pigment-protein complexes (PPCs). These parameters comprise (i) the local transition energies of the pigments in their binding sites in the protein, the site energies; (ii) the couplings between optical transitions of the pigments, the excitonic couplings; and (iii) the spectral density characterizing the dynamic modulation of pigment transition energies and excitonic couplings by protein vibrations. Starting with quantum mechanics perturbation theory, we provide a microscopic foundation for the standard PPC Hamiltonian and relate the expressions obtained for its matrix elements to quantities that can be calculated with classical molecular mechanics/electrostatics approaches including the whole PPC in atomic detail and using charge and transition densities obtained with quantum chemical calculations on the isolated building blocks of the PPC. In the second part of this perspective, the Hamiltonian is utilized to describe the quantum dynamics of excitons. Situations are discussed that differ in the relative strength of excitonic and exciton-vibrational coupling. The predictive power of the approaches is demonstrated in application to different PPCs, and challenges for future work are outlined.

Refined definition of the critical micelle concentration and application to alkyl maltosides used in membrane protein research.

The critical micelle concentration (CMC) of nonionic detergents is defined as the breaking point in the monomer concentration as a function of the total detergent concentration, identified by setting the third derivate of this function to zero. Combined with a mass action model for micelle formation, this definition yields analytic formulae for the concentration ratio of monomers to total detergent at the CMC and the relationship between the CMC and the free energy of micellization g mic. The theoretical breaking point is shown to coincide with the breaking point of the experimental titration curve, if the fluorescence enhancement of 8-anilino-1-naphthalene-sulfonic acid (ANS) or a similar probe dye is used to monitor micelle formation. Application to a series of n-alkyl-ß-d-maltosides with the number of carbon atoms in the alkyl chain ranging from 8 to 12 demonstrates the good performance of a molecular thermodynamic model, in which the free energy of micellization is given by g mic = σΦ + g pack + g st. In this model, σ is a fit parameter with the dimension of surface tension, Φ represents the change in area of hydrophobic molecular surfaces in contact with the aqueous phase, and g pack and g st are contributions, respectively, from alkyl chain packing in the micelle interior and steric repulsion of detergent head groups. The analysis of experimental data from different sources shows that varying experimental conditions such as co-solutes in the aqueous phase can be accounted for by adapting only σ, if the co-solutes do not bind to the detergent to an appreciable extent. The model is considered a good compromise between theory and practicability to be applied in the context of in vitro investigations of membrane proteins.

A unified framework for the numerical evaluation of the Q-subtractive Kramers-Kronig relations and application to the reconstruction of optical constants of quartz.

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, F1, is reconstructed using the imaginary part F2, obtained from an experiment. The focus is on multiply (Q-)subtractive KK relations, which in contrast to the standard KK transformation, exploit information about F1 at a certain number Q of anchor frequencies. We develop a general mathematical framework of the Q-subtractive KK relations and analyze all sources of errors contributing to the inaccuracy of the reconstructed F1. We show that for the reconstruction of F1 only a single evaluation of the standard KK relation is needed together with a correction term given by an approximate evaluation of the error in the standard KK. It is demonstrated that in the classical form of the Q-subtractive KK relations, this correction term coincides with the Lagrange interpolation polynomial of the error with nodes at the anchor frequencies. Another correction term can also be constructed as a lower degree polynomial through a least squares fit, a particular realization of which is taking the average of Q singly subtractive KK relations. As a result, recommendations for the application of Q-subtractive KK relations are given. The accuracy of the considered approaches is illustrated on synthetic examples and experimental data of fused SiO2.

Short-Range Effects in the Special Pair of Photosystem II Reaction Centers: The Nonconservative Nature of Circular Dichroism.

Photosystem II reaction centers extract electrons from water, providing the basis of oxygenic life on earth. Among the light-sensitive pigments of the reaction center, a central chlorophyll a dimer, known as the special pair, so far has escaped a complete theoretical characterization of its excited state properties. The close proximity of the special pair pigments gives rise to short-range effects that comprise a coupling between local and charge transfer (CT) excited states as well as other intermolecular quantum effects. Using a multiscale simulation and a diabatization technique, we show that the coupling to CT states is responsible for 45% of the excitonic coupling in the special pair. The other short-range effects cause a nonconservative nature of the circular dichroism spectrum of the reaction center by effectively rotating the electric transition dipole moments of the special pair pigments inverting and strongly enhancing their intrinsic rotational strength.

Structural basis of cyanobacterial photosystem II Inhibition by the herbicide terbutryn.

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.