The effect of hydration on protein flexibility in photosystem II of green plants studied by quasielastic neutron scattering

The effect of hydration on protein dynamics in photosystem II (PS II) membrane fragments from spinach has been investigated by using the method of quasielastic neutron scattering (QENS) at room temperature. The QENS data obtained indicate that the protein dynamics is strongly dependent on the extent of hydration. In particular, the hydration-induced activation of localized diffusive protein motions and Q_A⁻ reoxidation by Q_B in PS II appear to be correlated in their onset at a hydration value of about 45% relative humidity (r.h.). These findings underline the crucial functional relevance of localized diffusive protein motions on the picosecond-timescale for the reactions of light-induced photosynthetic water splitting under formation of plastoquinol and molecular oxygen in PS II of green plants. Advanced neutron scattering and complementary techniques to study biological systems. Contributions from the meetings, “Neutrons in Biology”, STFC Rutherford Appleton Laboratory, Didcot, UK, 11–13 July and “Proteins At Work 2007”, Perugia, Italy, 28–30 May 2007.     

Using polarization analysis to separate the coherent and incoherent scattering from protein samples

Polarization analysis was used to separate experimentally the coherent and spin-incoherent nuclear static scattering functions, from a representative set of samples of interest for protein studies. This method had so far limited application in the study of amorphous materials, despite the relevance of the information that it provides. It allows, for instance, the experimental determination of the structure factor of materials containing a significant amount of hydrogen atoms, avoiding the contamination of measurements by a non-negligible incoherent background. Knowledge of the relative importance of the coherent and incoherent terms at different Q-values is also a pre-requisite for the interpretation of quasielastic neutron scattering experiments, performed at instruments in which the total dynamic scattering function is measured, such as conventional time-of-flight and backscattering spectrometers. Combining data from different instruments, it was possible to cover a wide Q-range, from the small-angle region (0.006 < Q < 0.04 Å^− 1) to the wide-angle region (up to ≈ 2.35 Å^− 1). Quantitative information was obtained on the fraction of coherent to spin-incoherent scattering from different protein samples: deuterated and protonated protein powders at different hydration levels and solutions of protonated proteins in D₂O at different concentrations. The results obtained are discussed in the context of the validity of the assumptions generally made when interpreting quasielastic neutron scattering experiments performed without polarization analysis.     

Excitation dynamics of two spectral forms of the core complexes from photosynthetic bacterium Thermochromatium tepidum

The intact core antenna-reaction center (LH1-RC) core complex of thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum is peculiar in its long-wavelength LH1-Q_y absorption (915 nm). We have attempted comparative studies on the excitation dynamics of bacteriochlorophyll (BChl) and carotenoid (Car) between the intact core complex and the EDTA-treated one with the Q_y absorption at 889 nm. For both spectral forms, the overall Car-to-BChl excitation energy transfer efficiency is determined to be ∼20%, which is considerably lower than the reported values, e.g., ∼35%, for other photosynthetic purple bacteria containing the same kind of Car (spirilloxanthin). The RC trapping time constants are found to be 50∼60 ps (170∼200 ps) for RC in open (closed) state irrespective to the spectral forms and the wavelengths of Q_y excitation. Despite the low-energy LH1-Q_y absorption, the RC trapping time are comparable to those reported for other photosynthetic bacteria with normal LH1-Q_y absorption at 880 nm. Selective excitation to Car results in distinct differences in the Q_y-bleaching dynamics between the two different spectral forms. This, together with the Car band-shift signals in response to Q_y excitation, reveals the presence of two major groups of BChls in the LH1 of Tch. tepidum with a spectral heterogeneity of ∼240 cm⁻¹, as well as an alteration in BChl-Car geometry in the 889-nm preparation with respect to the native one.     

Phycocyanin sensitizes both photosystem I and photosystem II in cryptophyte Chroomonas CCMP270 cells

This article presents an investigation of the energy migration dynamics in intact cells of the unicellular photosynthetic cryptophyte Chroomonas CCMP270 by steady-state and time-resolved fluorescence measurements. By kinetic modeling of the fluorescence data on chlorophyll and phycocyanin 645 excitation (at 400 and 582 nm respectively), it has been possible to show the excited state energy distribution in the photosynthetic antenna of this alga. Excitation energy from phycocyanin 645 is distributed nearly equally between photosystem I and photosystem II with very high efficiency on a 100-ps timescale. The excitation energy trapping times for both photosystem I (∼30 ps) and photosystem I (200 and ∼540 ps) correspond well to those obtained from experiments on isolated photosystems. The results are compared with previous results for another cryptophyte species, Rhodomonas CS24, and suggest a similar membrane organization for the cryptophytes with the phycobiliproteins tightly packed in the thylakoid lumen around the periphery of the photosystems.     

Low-temperature molecular dynamics simulations of horse heart cytochrome c and comparison with inelastic neutron scattering data

Molecular dynamics (MD) simulation combined with inelastic neutron scattering can provide information about the thermal dynamics of proteins, especially the low-frequency vibrational modes responsible for large movement of some parts of protein molecules. We performed several 30-ns MD simulations of cytochrome c (Cyt c) in a water box for temperatures ranging from 110 to 300 K and compared the results with those from experimental inelastic neutron scattering. The low-frequency vibrational modes were obtained via dynamic structure factors, S(Q, ω), obtained both from inelastic neutron scattering experiments and calculated from MD simulations for Cyt c in the same range of temperatures. The well known thermal transition in structural movements of Cyt c is clearly seen in MD simulations; it is, however, confined to unstructured fragments of loops Ω₁ and Ω₂; movement of structured loop Ω₃ and both helical ends of the protein is resistant to thermal disturbance. Calculated and experimental S(Q, ω) plots are in qualitative agreement for low temperatures whereas above 200 K a boson peak vanishes from the calculated plots. This may be a result of loss of crystal structure by the protein–water system compared with the protein crystal.     

Photosynthetic electron transport activity in heat-treated barley leaves: The role of internal alternative electron donors to photosystem II

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 °C, 40 s). Under these circumstances, the K peak (∼ F_400 μs) appears in the chl a fluorescence (OJIP) transient reflecting partial Q_A reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q_A⁻ accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q_A⁻ accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t_1/2 ∼ 30 ms. This alternative electron donor is most probably ascorbate.     

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.     

Identification of the Intermediate Charge-Separated State PβL in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B_L and B_M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm⁻¹ and 1770 cm⁻¹, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P^∗ decayed on a timescale of ∼6.3 ps to P⁺B_L⁻. The decay of P⁺B_L⁻ occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β_L BChl resulted in formation of the P⁺β_L⁻ state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P⁺B_L⁻ and P⁺β_L⁻ showed clear differences related to the different environments of the two BChls in the mutant RC.     

In vivo measurement of internal and global macromolecular motions in Escherichia coli

We present direct quasielastic neutron scattering measurements, in vivo, of macromolecular dynamics in Escherichia coli. The experiments were performed on a wide range of timescales to cover the large panel of internal and self-diffusion motions. Three major internal processes were extracted at physiological temperature: a fast picosecond process that corresponded to restricted jump diffusion motions and two slower processes that resulted from reorientational motions occurring in ∼40 ps and 90 ps, respectively. The analysis of the fast process revealed that the cellular environment leads to an appreciable increase in internal molecular flexibility and diffusive motion rates compared with those evaluated in fully hydrated powders. The result showed that the amount of cell water plays a decisive role in internal molecular dynamics. Macromolecular interactions and confinement, however, attenuate slightly the lubricating effect of water, as revealed by the decrease of the in vivo parameters compared with those measured in solution. The study demonstrated that standard sample preparations do not mimic accurately the physiological environment and suggested that intracellular complexity participates in functional dynamics necessary for biological activity. Furthermore, the method allowed the extraction of the self-diffusion of E. coli macromolecules, which presented similar parameters as those extracted for hemoglobin in red blood cells.     

Reaction pattern of Photosystem II: oxidative water cleavage and protein flexibility

This short communication addresses three topics of photosynthetic water cleavage in Photosystem II (PS II): (a) effect of protonation in the acidic range on the extent of the ‘fast’ ns kinetics of P680^+· reduction by Y_Z, (b) mechanism of O–O bond formation and (c) role of protein flexibility in the functional integrity of PS II. Based on measurements of light-induced absorption changes and quasielastic neutron scattering in combination with mechanistic considerations, evidence is presented for the protein acting as a functionally active constituent of the water cleavage machinery, in particular, for directed local proton transfer. A specific flexibility emerging above a threshold of about 230 K is an indispensable prerequisite for oxygen evolution and plastoquinol formation.     

Carboxylate shifts steer interquinone electron transfer in photosynthesis

Understanding the mechanisms of electron transfer (ET) in photosynthetic reaction centers (RCs) may inspire novel catalysts for sunlight-driven fuel production. The electron exit pathway of type II RCs comprises two quinone molecules working in series and in between a non-heme iron atom with a carboxyl ligand (bicarbonate in photosystem II (PSII), glutamate in bacterial RCs). For decades, the functional role of the iron has remained enigmatic. We tracked the iron site using microsecond-resolution x-ray absorption spectroscopy after laser-flash excitation of PSII. After formation of the reduced primary quinone, Q_A⁻, the x-ray spectral changes revealed a transition (t_½ ≈ 150 μs) from a bidentate to a monodentate coordination of the bicarbonate at the Fe(II) (carboxylate shift), which reverted concomitantly with the slower ET to the secondary quinone Q_B. A redox change of the iron during the ET was excluded. Density-functional theory calculations corroborated the carboxylate shift both in PSII and bacterial RCs and disclosed underlying changes in electronic configuration. We propose that the iron-carboxyl complex facilitates the first interquinone ET by optimizing charge distribution and hydrogen bonding within the Q_AFeQ_B triad for high yield Q_B reduction. Formation of a specific priming intermediate by nuclear rearrangements, setting the stage for subsequent ET, may be a common motif in reactions of biological redox cofactors.     

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: Characterization of the rbcL deletion mutant of tobacco

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q_A and Q_B, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F₀ ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O₂ in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.     

Sites of inhibition by disulfiram in thylakoid membranes

Disulfiram (tetraethylthiuram disulfide), a metal chelator, inhibits photosynthetic electron transport in broken chloroplasts. A major site of inhibition is detected on the electron-acceptor side of photosystem II between Q_A, the first plastoquinone electron-acceptor, and the second plastoquinone electron-acceptor, Q_B. This site of inhibition is shown by a severalfold increase in the half-time of Q⁻_A oxidation, as monitored by the decay of the variable chlorophyll a flourescence after an actinic flash. Another site of inhibition is detected in the functioning of the reaction center of photosystem II; disulfiram is observed to quench the room temperature variable chlorophyll a fluorescence, as well as the intensity of the 695 nm peak, relative to the 685 nm peak, in the chlorophyll a fluorescence spectrum at 77 K. Electron transport from H₂O to the photosystem II electron-acceptor silicomolybdate is also inhibited. Disulfiram does not inhibit electron flow before the site(s) of donation by exogenous electron donors to photosystem II, and no inhibition is detected in the partial reactions associated with photosystem I.     

Kinetics of photosystem II electron transport: a mathematical analysis based on chlorophyll fluorescence induction

The OJDIP rise in chlorophyll fluorescence during induction at different light intensities was mathematically modeled using 24 master equations describing electron transport through photosystem II (PSII) plus ordinary differential equations for electron budgets in plastoquinone, cytochrome f, plastocyanin, photosystem I, and ferredoxin. A novel feature of the model is consideration of electron in- and outflow budgets resulting in changes in redox states of Tyrosine Z, P680, and Q_A as sole bases for changes in fluorescence yield during the transient. Ad hoc contributions by transmembrane electric fields, protein conformational changes, or other putative quenching species were unnecessary to account for primary features of the phenomenon, except a peculiar slowdown of intra-PSII electron transport during induction at low light intensities. The lower than F _m post-flash fluorescence yield F _f was related to oxidized tyrosine Z. The transient J peak was associated with equal rates of electron arrival to and departure from Q_A and requires that electron transfer from Q_A ^? to Q_B be slower than that from Q_A ^? to Q_B ^?. Strong quenching by oxidized P680 caused the dip D. Reduced plastoquinone, a competitive product inhibitor of PSII, blocked electron transport proportionally with its concentration. Electron transport rate indicated by fluorescence quenching was faster than the rate indicated by O₂ evolution, because oxidized donor side carriers quench fluorescence but do not transport electrons. The thermal phase of the fluorescence rise beyond the J phase was caused by a progressive increase in the fraction of PSII with reduced Q_A and reduced donor side.     

Hydration effect on low-frequency protein dynamics observed in simulated neutron scattering spectra

Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (<∼20 μeV) is developed in the near future.     

Modification of energy-transfer processes in the cyanobacterium, Arthrospira platensis, to adapt to light conditions, probed by time-resolved fluorescence spectroscopy

In cyanobacteria, the interactions among pigment–protein complexes are modified in response to changes in light conditions. In the present study, we analyzed excitation energy transfer from the phycobilisome and photosystem II to photosystem I in the cyanobacterium Arthrospira (Spirulina) platensis. The cells were grown under lights with different spectral profiles and under different light intensities, and the energy-transfer characteristics were evaluated using steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopy techniques. The fluorescence rise and decay curves were analyzed by global analysis to obtain fluorescence decay-associated spectra. The direct energy transfer from the phycobilisome to photosystem I and energy transfer from photosystem II to photosystem I were modified depending on the light quality, light quantity, and cultivation period. However, the total amount of energy transferred to photosystem I remained constant under the different growth conditions. We discuss the differences in energy-transfer processes under different cultivation and light conditions.     

Acclimation of photosynthesis to nitrogen deficiency in Phaseolus vulgaris

Nitrogen deficiency diminishes consumption of photosynthates in anabolic metabolism. We studied adjustments of the photosynthetic machinery in nitrogen-deficient bean plants and found four phenomena. First, the number of chloroplasts per cell decreased. Chloroplasts of nitrogen starved leaves contained less pigments than those of control leaves, but the in vitro activities of light reactions did not change when measured on chlorophyll basis. Second, nitrogen deficiency induced cyclic electron transfer. The amounts of Rubisco and ferredoxin-NADP⁺ reductase decreased in nitrogen starved plants. Low activities of these enzymes are expected to lead to increase in reduction of oxygen by photosystem I. However, diaminobenzidine staining did not reveal hydrogen peroxide production in nitrogen starved plants. Measurements of far-red-light-induced redox changes of the primary donor of photosystem I suggested that instead of producing oxygen radicals, nitrogen starved plants develop a high activity of cyclic electron transport that competes with oxygen for electrons. Nitrogen starvation led to decrease in photochemical quenching and increase in non-photochemical quenching, indicating that cyclic electron transport reduces the plastoquinone pool and acidifies the lumen. A third effect is redistribution of excitation energy between the photosystems in favor of photosystem I. Thus, thylakoids of nitrogen starved plants appeared to be locked in state 2, which further protects photosystem II by decreasing its absorption cross-section. As a fourth response, the proportion of non-Q_B-reducing photosystem II reaction centers increased and the redox potential of the Q_B/Q_B⁻ pair decreased by 25 mV in a fraction of photosystem II centers of nitrogen starved plants.