Impact of Peripheral Hydrogen Bond on Electronic Properties of the Primary Acceptor Chlorophyll in the Reaction Center of Photosystem I.

Photosystem I (PS I) is a photosynthetic pigment-protein complex that absorbs light and uses the absorbed energy to initiate electron transfer. Electron transfer has been shown to occur concurrently along two (A- and B-) branches of reaction center (RC) cofactors. The electron transfer chain originates from a special pair of chlorophyll a molecules (P700), followed by two chlorophylls and one phylloquinone in each branch (denoted as A-1, A0, A1, respectively), converging in a single iron-sulfur complex Fx. While there is a consensus that the ultimate electron donor-acceptor pair is P700+A0-, the involvement of A-1 in electron transfer, as well as the mechanism of the very first step in the charge separation sequence, has been under debate. To resolve this question, multiple groups have targeted electron transfer cofactors by site-directed mutations. In this work, the peripheral hydrogen bonds to keto groups of A0 chlorophylls have been disrupted by mutagenesis. Four mutants were generated: PsaA-Y692F; PsaB-Y667F; PsaB-Y667A; and a double mutant PsaA-Y692F/PsaB-Y667F. Contrary to expectations, but in agreement with density functional theory modeling, the removal of the hydrogen bond by Tyr → Phe substitution was found to have a negligible effect on redox potentials and optical absorption spectra of respective chlorophylls. In contrast, Tyr → Ala substitution was shown to have a fatal effect on the PS I function. It is thus inferred that PsaA-Y692 and PsaB-Y667 residues have primarily structural significance, and their ability to coordinate respective chlorophylls in electron transfer via hydrogen bond plays a minor role.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex.

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl ß-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

Copper(I)-Pyrazolate Complexes as Solid-State Phosphors: Deep-Blue Emission through a Remote Steric Effect.

We describe a novel manifestation of rigidochromic behavior in a series of tetranuclear Cu(I)-pyrazolate (Cu4pz4) macrocycles, with implications for solid-state luminescence at deep-blue wavelengths (<460 nm). The Cu4pz4 emissions are remarkably sensitive to structural effects far from the luminescent core: when 3,5-di-tert-butylpyrazoles are used as bridging ligands, adding a C4 substituent can induce a blue shift of more than 100 nm. X-ray crystal and computational analyses reveal that C4 units influence the conformational behavior of adjacent tert-butyl groups, with a subsequent impact on the global conformation of the Cu4pz4 complex. Emissions are mediated primarily through a cluster-centered triplet (3CC) state; compression of the Cu4 cluster into a nearly close-packed geometry prevents the reorganization of its excited-state structure and preserves the 3CC energy at a high level. The remote steric effect may thus offer alternative strategies toward the design of phosphors with rigid excited-state geometries.

Conserved residue PsaB-Trp673 is essential for high-efficiency electron transfer between the phylloquinones and the iron-sulfur clusters in Photosystem I.

Despite the high level of symmetry between the PsaA and PsaB polypeptides in Photosystem I, some amino acids pairs are strikingly different, such as PsaA-Gly693 and PsaB-Trp673, which are located near a cluster of 11 water molecules between the A1A and A1B quinones and the FX iron-sulfur cluster. In this work, we changed PsaB-Trp673 to PsaB-Phe673 in Synechocystis sp. PCC 6803. The variant contains ~ 85% of wild-type (WT) levels of Photosystem I but is unable to grow photoautotrophically. Both time-resolved and steady-state optical measurements show that in the PsaB-W673F variant less than 50% of the electrons reach the terminal iron-sulfur clusters FA and FB; the majority of the electrons recombine from A1A- and A1B-. However, in those reaction centers which pass electrons forward the transfer is heterogeneous: a minor population shows electron transfer rates from A1A- and A1B- to FX slightly slower than that of the WT, whereas a major population shows forward electron transfer rates to FX slowed to the ~ 10 µs time range. Competition between relatively similar forward and backward rates of electron transfer from the quinones to the FX cluster account for the relatively low yield of long-lived charge separation in the PsaB-W673F variant. A higher water content and its increased mobility observed in MD simulations in the interquinone cavity of the PsaB-W673F variant shifts the pK of PsaB-Asp575 and allows its deprotonation in situ. The heterogeneity found may be rooted in protonation state of PsaB-Asp575, which controls whether electron transfer can proceed beyond the phylloquinone cofactors.

Triplet-Triplet Coupling in Chromophore Dimers: Theory and Experiment.

Knowledge of triplet state energies and triplet-triplet (T-T) interactions in aggregated organic molecules is essential for understanding photochemistry and dynamics of many natural and artificial systems. In this work, we combine direct phosphorescence measurements of triplet state energies, which are challenging due to the spin-forbidden nature of respective transitions and applicable to only a limited number of systems, with quantum chemical computational tools that can provide valuable qualitative and quantitative information about triplet states of interacting molecules. Using hexatriene, protoporphyrin, pheophorbide, and chlorophyll dimers as model systems, we demonstrate a complicated dependence of T-T coupling on a relative orientation of chromophores, governed by a nodal structure of overlapping electronic wave functions, that modulates interpigment interactions by orders of magnitude. It is also shown that geometrical relaxation of the triplet state is one of the critical factors for predictive modeling of T-T interactions in molecular aggregates.

Probing the excitonic landscape of the Chlorobaculum tepidum Fenna-Matthews-Olson (FMO) complex: a mutagenesis approach.

In this paper we report the steady-state optical properties of a series of site-directed mutants in the Fenna-Matthews-Olson (FMO) complex of Chlorobaculum tepidum, a photosynthetic green sulfur bacterium. The FMO antenna complex has historically been used as a model system for energy transfer due to the water-soluble nature of the protein, its stability at room temperature, as well as the availability of high-resolution structural data. Eight FMO mutants were constructed with changes in the environment of each of the bacteriochlorophyll a pigments found within each monomer of the homotrimeric FMO complex. Our results reveal multiple changes in low temperature absorption, as well as room temperature CD in each mutant compared to the wild-type FMO complex. These datasets were subsequently used to model the site energies of each pigment in the FMO complex by employing three different Hamiltonians from the literature. This enabled a basic approximation of the site energy shifts imparted on each pigment by the changed amino acid residue. These simulations suggest that, while the three Hamiltonians used in this work provide good fits to the wild-type FMO absorption spectrum, further efforts are required to obtain good fits to the mutant minus wild-type absorption difference spectra. This demonstrates that the use of FMO mutants can be a valuable tool to refine and iterate the current models of energy transfer in this system.

Oxygen concentration inside a functioning photosynthetic cell.

The excess oxygen concentration in the photosynthetic membranes of functioning oxygenic photosynthetic cells was estimated using classical diffusion theory combined with experimental data on oxygen production rates of cyanobacterial cells. The excess oxygen concentration within the plesiomorphic cyanobacterium Gloeobactor violaceus is only 0.025 µM, or four orders of magnitude lower than the oxygen concentration in air-saturated water. Such a low concentration suggests that the first oxygenic photosynthetic bacteria in solitary form could have evolved ∼2.8 billion years ago without special mechanisms to protect them against reactive oxygen species. These mechanisms instead could have been developed during the following ∼500 million years while the oxygen level in the Earth's atmosphere was slowly rising. Excess oxygen concentrations within individual cells of the apomorphic cyanobacteria Synechocystis and Synechococcus are 0.064 and 0.25 µM, respectively. These numbers suggest that intramembrane and intracellular proteins in isolated oxygenic photosynthetic cells are not subjected to excessively high oxygen levels. The situation is different for closely packed colonies of photosynthetic cells. Calculations show that the excess concentration within colonies that are ∼40 µm or larger in diameter can be comparable to the oxygen concentration in air-saturated water, suggesting that species forming colonies require protection against reactive oxygen species even in the absence of oxygen in the surrounding atmosphere.

Singlet exciton fission in polycrystalline thin films of a slip-stacked perylenediimide.

The crystal structure of N,N-bis(n-octyl)-2,5,8,11-tetraphenylperylene-3,4:9,10-bis(dicarboximide), 1, obtained by X-ray diffraction reveals that 1 has a nearly planar perylene core and π-π stacks at a 3.5 Å interplanar distance in well-separated slip-stacked columns. Theory predicts that slip-stacked, π-π-stacked structures should enhance interchromophore electronic coupling and thus favor singlet exciton fission. Photoexcitation of vapor-deposited polycrystalline 188 nm thick films of 1 results in a 140 ± 20% yield of triplet excitons ((3*)1) in τ(SF) = 180 ± 10 ps. These results illustrate a design strategy for producing perylenediimide and related rylene derivatives that have the optimized interchromophore electronic interactions which promote high-yield singlet exciton fission for potentially enhancing organic solar cell performance and charge separation in systems for artificial photosynthesis.

Temporal and spectral characterization of the photosynthetic reaction center from Heliobacterium modesticaldum.

A time-resolved spectroscopic study of the isolated photosynthetic reaction center (RC) from Heliobacterium modesticaldum reveals that thermal equilibration of light excitation among the antenna pigments followed by trapping of excitation and the formation of the charge-separated state P800 (+)A0 (-) occurs within ~25 ps. This time scale is similar to that reported for plant and cyanobacterial photosystem I (PS I) complexes. Subsequent electron transfer from the primary electron acceptor A0 occurs with a lifetime of ~600 ps, suggesting that the RC of H. modesticaldum is functionally similar to that of Heliobacillus mobilis and Heliobacterium chlorum. The (A0 (-) - A0) and (P800 (+) - P800) absorption difference spectra imply that an 8(1)-OH-Chl a F molecule serves as the primary electron acceptor and occupies the position analogous to ec3 (A0) in PS I, while a monomeric BChl g pigment occupies the position analogous to ec2 (accessory Chl). The presence of an intense photobleaching band at 790 nm in the (A0 (-) - A0) spectrum suggests that the excitonic coupling between the monomeric accessory BChl g and the 8(1)-OH-Chl a F in the heliobacterial RC is significantly stronger than the excitonic coupling between the equivalent pigments in PS I.

Photoexcitation of Fe3 O Nodes in MOF Drives Water Oxidation at pH=1 When Ru Catalyst Is Present.

Artificial photosynthesis strives to convert the energy of sunlight into sustainable, eco-friendly solar fuels. However, systems with light-driven water oxidation reaction (WOR) at pH=1 are rare. Broadly used [Ru(bpy)3 ]2+ (bpy=2,2'-bipyridine) photosensitizer has a fixed +1.23 V potential which is insufficient to drive most water oxidation catalysts (WOCs) in acid, while Fe2 O3 , featuring the highly oxidizing holes, is not stable at low pH. Here, the key examples of Fe-based metal-organic framework (MOF) water oxidation photoelectrocatalysts active at pH=1 are presented. Fe-MIL-126 and Fe MOF-dcbpy structures were formed with 4,4'-biphenyl dicarboxylate (bpdc), 2,2'-bipyridine-5,5'-dicarboxylate (dcbpy) linkers and their mixtures. Presence of dcbpy linkers allows integration of metal-based catalysts via coordination to 2,2'-bipyridine fragments. Fe-based MOFs were doped with Ru-based precursors to achieve highly active MOFs bearing [Ru(bpy)(dcbpy)(H2 O)2 ]2+ WOC. Materials were analyzed with X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infra-red (FTIR) spectroscopy, resonance Raman, X-ray absorption spectroscopy, fs optical pump-probe, electron paramagnetic resonance (EPR), diffuse reflectance and electric conductivity measurements and were modeled by band structure calculations. It is shown that under reaction conditions, FeIII and RuIII oxidation states are present, indicating rate-limiting electron transfer in MOF. Fe3 O nodes emerge as photosensitizers able to drive prolonged O2 evolution in acid. Further developments are possible via MOF's linker modification for enhanced light absorption, electrical conductivity, reduced MOF solubility in acid, Ru-WOC modification for faster WOC catalysis, or Ru-WOC substitution to 3d metal-based systems. The findings give further insight for development of light-driven water splitting systems based on Earth-abundant metals.

Predicting Mutation-Induced Changes in the Electronic Properties of Photosynthetic Proteins from First Principles: The Fenna-Matthews-Olson Complex Example.

Multiscale molecular modeling is utilized to predict optical absorption and circular dichroism spectra of two single-point mutants of the Fenna-Matthews-Olson photosynthetic pigment-protein complex. The modeling approach combines classical molecular dynamics simulations with structural refinement of photosynthetic pigments and calculations of their excited states in a polarizable protein environment. The only experimental input to the modeling protocol is the X-ray structure of the wild-type protein. The first-principles modeling reproduces changes in the experimental optical spectra of the considered mutants, Y16F and Q198V. Interestingly, the Q198V mutation has a negligible effect on the electronic properties of the targeted bacteriochlorophyll a pigment. Instead, the electronic properties of several other pigments respond to this mutation. The molecular modeling demonstrates that a single-point mutation can induce long-range effects on the protein structure, while extensive structural changes near a pigment do not necessarily lead to significant changes in the electronic properties of that pigment.

Predictive First-Principles Modeling of a Photosynthetic Antenna Protein: The Fenna-Matthews-Olson Complex.

High efficiency of light harvesting in photosynthetic pigment-protein complexes is governed by evolutionary-perfected protein-assisted tuning of individual pigment properties and interpigment interactions. Due to the large number of spectrally overlapping pigments in a typical photosynthetic complex, experimental methods often fail to unambiguously identify individual chromophore properties. Here, we report a first-principles-based modeling protocol capable of predicting properties of pigments in protein environment to a high precision. The technique was applied to successfully uncover electronic properties of the Fenna-Matthews-Olson (FMO) pigment-protein complex. Each of the three subunits of the FMO complex contains eight strongly coupled bacteriochlorophyll a (BChl a) pigments. The excitonic structure of FMO can be described by an electronic Hamiltonian containing excitation (site) energies of BChl a pigments and electronic couplings between them. Several such Hamiltonians have been developed in the past based on the information from various spectroscopic measurements of FMO; however, fine details of the excitonic structure and energy transfer in FMO, especially assignments of short-lived high-energy sites, remain elusive. Utilizing polarizable embedding quantum mechanics/molecular mechanics with the effective fragment potentials, we computed the electronic Hamiltonian of FMO that is in general agreement with previously reported empirical Hamiltonians and quantitatively reproduces experimental absorption and circular dichroism spectra of the FMO protein. The developed computational protocol is sufficiently simple and can be utilized for predictive modeling of other wild-type and mutated photosynthetic pigment-protein complexes.

Near shot-noise limited time-resolved circular dichroism pump-probe spectrometer.

We describe an optical near shot-noise limited time-resolved circular dichroism (TRCD) pump-probe spectrometer capable of reliably measuring circular dichroism signals in the order of µdeg with nanosecond time resolution. Such sensitivity is achieved through a modification of existing TRCD designs and introduction of a new data processing protocol that eliminates approximations that have caused substantial nonlinearities in past measurements and allows the measurement of absorption and circular dichroism transients simultaneously with a single pump pulse. The exceptional signal-to-noise ratio of the described setup makes the TRCD technique applicable to a large range of non-biological and biological systems. The spectrometer was used to record, for the first time, weak TRCD kinetics associated with the triplet state energy transfer in the photosynthetic Fenna-Matthews-Olson antenna pigment-protein complex.

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 that eliminates the risk of a false negative signal.

A DNA hybridization based optical detection platform for the detection of foodborne pathogens has been developed with virtually zero probability of the false negative signal. This portable, low-cost and real-time assaying detection platform utilizes the color changing molecular beacon as a probe for the optical detection of the target sequence. The computer-controlled detection platform exploits the target hybridization induced change of fluorescence color due to the Förster (fluorescence) resonance energy transfer (FRET) between a pair of spectrally shifted fluorophores conjugated to the opposite ends of a beacon (oligonucleotide probe). Unlike the traditional fluorophore-quencher beacon design, the presence of two fluorescence molecules allows to actively visualize both hybridized and unhybridized states of the beacon. This eliminates false negative signal detection characteristic for the fluorophore-quencher beacon where bleaching of the fluorophore or washout of a beacon is indistinguishable from the absence of the target DNA sequence. In perspective, the two-color design allows also to quantify the concentration of the target DNA in a sample down to < =1 ng/microl. The new design is suitable for simultaneous reliable detection of hundreds of DNA target sequences in one test run using a series of beacons immobilized on a single substrate in a spatial format.

Ultrafast optical pump-probe studies of the cytochrome b(6)f complex in solution and crystalline states.

The cytochrome b6f complex of oxygenic photosynthesis contains a single chlorophyll a (Chl a) molecule whose function is presently unknown. The singlet excited state of the Chl a molecule is quenched by the surrounding protein matrix, and thus the Chl a molecule in the b6f complex may serve as an exceptionally sensitive probe of the protein structure. For the first time, singlet excited-state dynamics were measured in well-diffracting crystals using femtosecond time-resolved optical pump-probe methodology. Lifetimes of the Chl a molecule in crystals of the cytochrome b6f complex having different space groups were 3-6 times longer than those determined in detergent solutions of the b6f. The observed differences in excited state dynamics may arise from small (1-1.5 A) changes in the local protein structure caused by crystal packing. The Chl a excited state lifetimes measured in the dissolved cytochrome b6f complexes from several different species are essentially the same, in spite of differences in the local amino acid sequences around the Chl a. This supports an earlier hypothesis that the short excited state lifetime of Chl a is critical for the function of the b6f complex.

Does the singlet minus triplet spectrum with major photobleaching band near 680-682 nm represent an intact reaction center of Photosystem II?

We use both frequency- and time-domain low-temperature (5-20 K) spectroscopies to further elucidate the shape and spectral position of singlet minus triplet (triplet-bottleneck) spectra in the reaction centers (RCs) of Photosystem II (PSII) isolated from wild-type Chlamydomonas reinhardtii and spinach. It is shown that the shape of the nonresonant transient hole-burned spectrum in destabilized RCs from C. reinhardtii is very similar to that typically observed for spinach. This suggests that the previously observed difference in transient spectra between RCs from C. reinhardtii and spinach is not due to the sample origin but most likely due to a partial destabilization of the D1 and D2 polypeptides. This supports our previous assignments that destabilized RCs (referred to as RC680) (Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870), with a major photobleaching band near 680-682 nm and the absence of a photobleaching band near 673 nm, do not represent the intact RC residing within the PSII core complex. Time-resolved absorption difference spectra obtained for partially destabilized RCs of C. reinhardtii and for typical spinach RCs support the above conclusions. The absence of clear photobleaching bands near 673 and 684 nm (where the PD1 chlorophyll and the active pheophytin (PheoD1) contribute, respectively) in picosecond transient absorption spectra in both RCs studied in this work indicates that the cation can move from the primary electron donor (ChlD1) to PD1 (i.e., PD1ChlD1(+)PheoD1(-) → PD1(+)ChlD1PheoD1(-)). Therefore, we suggest that ChlD1 is the major electron donor in usually studied destabilized RCs (with a major photobleaching near 680-682 nm), although the PD1 path (where PD1 serves as the primary electron donor) is likely present in intact RCs, as discussed in Acharya, K. et al. J. Phys. Chem. B 2012, 116, 4860-4870.

The fate of the triplet excitations in the Fenna-Matthews-Olson complex.

The fate of triplet excited states in the Fenna-Matthew-Olson (FMO) pigment-protein complex is studied by means of time-resolved nanosecond spectroscopy and exciton model simulations. Experiments reveal microsecond triplet excited-state energy transfer between the bacteriochlorophyll (BChl) pigments, but show no evidence of triplet energy transfer to molecular oxygen, which is known to produce highly reactive singlet oxygen and is the leading cause of photo damage in photosynthetic proteins. The FMO complex is exceptionally photo stable despite the fact it contains no carotenoids, which could effectively quench triplet excited states of (bacterio)chlorophylls and are usually found within pigment-protein complexes. It is inferred that the triplet excitation is transferred to the lowest energy pigment, BChl 3, within the FMO complex, whose triplet state energy is shifted by pigment-protein interactions below that of the singlet oxygen excitation. Thus, the energy transfer to molecular oxygen is blocked and the FMO does not need carotenoids for photo protection.

Triplet excited state energies and phosphorescence spectra of (bacterio)chlorophylls.

(Bacterio)Chlorophyll ((B)Chl) molecules play a major role in photosynthetic light-harvesting proteins, and the knowledge of their triplet state energies is essential to understand the mechanisms of photodamage and photoprotection, as the triplet excitation energy of (B)Chl molecules can readily generate highly reactive singlet oxygen. The triplet state energies of 10 natural chlorophyll (Chl a, b, c2, d) and bacteriochlorophyll (BChl a, b, c, d, e, g) molecules and one bacteriopheophytin (BPheo g) have been directly determined via their phosphorescence spectra. Phosphorescence of four molecules (Chl c2, BChl e and g, BPheo g) was characterized for the first time. Additionally, the relative phosphorescence to fluorescence quantum yield for each molecule was determined. The measurements were performed at 77K using solvents providing a six-coordinate environment of the Mg(2+) ion, which allows direct comparison of these (B)Chls. Density functional calculations of the triplet state energies show good correlation with the experimentally determined energies. The correlation determined computationally was used to predict the triplet energies of three additional (B)Chl molecules: Chl c1, Chl f, and BChl f.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.