Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.     

Effect of deuteration and cryosolvents on the energy transduction in primary processes of photosynthesis.

Effects of cryosolvents and D2O/H2O substitution on the reaction centres (RCs) isolated from photosynthetic bacteria were studied with respect to the role of intra-protein hydrogen bonds in the primary photosynthetic electron transfer. As a result of such treatment of RCs, the charge separation rate between the photoactive bacteriochlorophyll (P2 dimer) and bacteriopheophytin and the rate of electron transfer to the primary quinone slowed down. The energy migration rate from bacteriopheophytin (BPheM), inactive in electron transport, to P2 decreased as well. Although cryosolvents can shift the redox potential of the photoactive pigment, there is no direct correlation between the P2 potential and the effects of these modifying agents on the photosynthetic process in RCs occurring with participation of P2. The removal of H subunit from the pigment-protein complex results in the pronounced weakening of the dimethyl sulfoxide modifying effects on the RC hydrogen bonds. The role of structural and dynamic state in the functioning of the photosynthetic bacterial RCs is analyzed. Relaxation processes in purple bacteria RCs accompanying the primary picosecond steps of energy transformation proceed with the participation of small proton-containing molecular groups in the immediate surroundings of electron transfer carriers. In this paper, we present results concerning mechanisms of primary photosynthetic steps, which were initiated by A. A. Krasnovsky and have been studied for several years at the Department of Biophysics. This paper is dedicated to the memory of our teacher Prof. A. A. Krasnovsky.     

Investigation of stability of photosynthetic reaction center and quantum dot hybrid films

The efficiency of interaction (efficiency of energy transfer) between various quantum dots (QDs) and photosynthetic reaction centers (RCs) from the purple bacterium Rhodobacter sphaeroides and conditions of long-term stability of functioning of such hybrid complexes in film preparations were investigated. It was found that dry films containing RCs and QDs and maintained at atmospheric humidity are capable to keep their functional activity for at least some months as judging by results of measurement of their spectral characteristics, efficiency of energy transfer from QDs to RCs, and RC electron-transport activity. Addition of trehalose to the films giving them still greater stability is especially expressed for films maintained at low humidity. These stable hybrid film structures are promising for further biotechnological studies for developing new phototransformation devices.     

Structural basis of bacterial photosynthetic reaction centers

The photosynthetic reaction center (RC) is the first membrane protein whose three-dimensional structure was revealed at the atomic level by X-ray crystallograph more than fifteen years ago. Structural information about RC made a great contribution to the understanding of the reaction mechanism of the complicated membrane protein complex. High-resolution structures of RCs from three photosynthetic bacteria are now available, namely, those from two mesophilic purple non-sulfur bacteria, Blastochloris viridis and Rhodobacter sphaeroides, and that from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. In addition, a variety of structural studies, mainly by X-ray crystallography, are still being performed to give more detailed insight into the reaction mechanism of this membrane protein. This review deals with structural studies of bacterial RC complexes, and a discussion about the electron transfer reaction between RCs and electron donors is the main focus out of several topics addressed by these structural studies. The structural data from three RCs and their electron donors provided reliable models for molecular recognition in the primary step of bacterial photosynthesis.     

Energy Transfer in Light-Adapted Photosynthetic Membranes: From Active to Saturated Photosynthesis

In bacterial photosynthesis light-harvesting complexes, LH2 and LH1 absorb sunlight energy and deliver it to reaction centers (RCs) with extraordinarily high efficiency. Submolecular resolution images have revealed that both the LH2:LH1 ratio, and the architecture of the photosynthetic membrane itself, adapt to light intensity. We investigate the functional implications of structural adaptations in the energy transfer performance in natural in vivo low- and high-light-adapted membrane architectures of Rhodospirillum photometricum. A model is presented to describe excitation migration across the full range of light intensities that cover states from active photosynthesis, where all RCs are available for charge separation, to saturated photosynthesis where all RCs are unavailable. Our study outlines three key findings. First, there is a critical light-energy density, below which the low-light adapted membrane is more efficient at absorbing photons and generating a charge separation at RCs, than the high-light-adapted membrane. Second, connectivity of core complexes is similar in both membranes, suggesting that, despite different growth conditions, a preferred transfer pathway is through core-core contacts. Third, there may be minimal subareas on the membrane which, containing the same LH2:LH1 ratio, behave as minimal functional units as far as excitation transfer efficiency is concerned.     

Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 micros. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA- QB/QA QB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA- X+/QA X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA- QB/QA QB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs.     

水深对苦草生长及叶片PSⅡ光化学特性的影响

利用植物效率仪(Handy-PEA)测定水深0.6、1.3、2.0 m下苦草叶片的快速荧光诱导动力学曲线(OJIP曲线),并采用JIP test方法分析和处理数据,研究水深对苦草生长和叶片光合机理的影响.结果表明: 随着水深增加,水下光强显著衰减,苦草生物量、无性系分株数、叶片数、根系总长度、根系表面积等形态指标显著降低,而最大叶长、平均叶长、最大叶宽无显著变化,2.0 m水深对苦草的生长产生了负面影响;单位反应中心吸收、捕获、电子传递、传递到电子传递末端的量子效率(ABS/RC、TR₀/RC、ET₀/RC、RE₀/RC)显著降低,单位反应中心的耗散量子效率(DI₀/RC)也显著下降,导致3种水深处理下单位反应中心吸收的能传递电子链末端的效率(φ_R0)以及用于电子传递的能量成功传递到电子链末端的效率(δ_R0)差异不显著,表明水深梯度对单位反应中心光合效率影响不显著;单位面积反应中心数(RC/CS₀)显著增加,相同受光面积时水深2.0 m处叶片光合作用显著强于水深0.6 m处;性能参数PI_abs、PI_cs和PI_abs,total显著提高,表明低光胁迫有利于光能向活跃化学能转变.苦草叶片通过激活未激活的反应中心,而不是提高单位反应中心光能利用效率来适应弱光强,且水深1.3 m较适合苦草生长.
     

水深对苦草生长及叶片PSⅡ光化学特性的影响

利用植物效率仪(Handy-PEA)测定水深0.6、1.3、2.0 m下苦草叶片的快速荧光诱导动力学曲线(OJIP曲线),并采用JIP test方法分析和处理数据,研究水深对苦草生长和叶片光合机理的影响.结果表明: 随着水深增加,水下光强显著衰减,苦草生物量、无性系分株数、叶片数、根系总长度、根系表面积等形态指标显著降低,而最大叶长、平均叶长、最大叶宽无显著变化,2.0 m水深对苦草的生长产生了负面影响;单位反应中心吸收、捕获、电子传递、传递到电子传递末端的量子效率(ABS/RC、TR₀/RC、ET₀/RC、RE₀/RC)显著降低,单位反应中心的耗散量子效率(DI₀/RC)也显著下降,导致3种水深处理下单位反应中心吸收的能传递电子链末端的效率(φ_R0)以及用于电子传递的能量成功传递到电子链末端的效率(δ_R0)差异不显著,表明水深梯度对单位反应中心光合效率影响不显著;单位面积反应中心数(RC/CS₀)显著增加,相同受光面积时水深2.0 m处叶片光合作用显著强于水深0.6 m处;性能参数PI_abs、PI_cs和PI_abs,total显著提高,表明低光胁迫有利于光能向活跃化学能转变.苦草叶片通过激活未激活的反应中心,而不是提高单位反应中心光能利用效率来适应弱光强,且水深1.3 m较适合苦草生长.
     

Nonlinear effect of dynamic self-organization in macromolecular systems caused by photocontrolled electron flux

We use the electron-conformational interaction approach to develop a physical model which self-consistently describes the photomobilized electron transfer kinetics and structure conformational transitions in reaction centers (RCs) of purple bacteria. We consider the kinetics of electron transition from pigment onto primary acceptor and the subsequent charge recombination accounting for the change of distance between the above-mentioned cofactors. It is shown that, given natural values of RC parameters, the kinetic constant's dependence on the acting light intensity is monotone. As opposed to the previous case, similar dependencies for the chain of electron transfer between primary and secondary quinone acceptors revealed anS-like relationship. This can lead to bistability of the RC optical transmission coefficient and a fundamental dependence of charge recombination kinetics upon the prehistory of the RC's interaction with exciting radiation.     

水稻光合日变化及光抑制的叶绿素荧光

研究了光敏核不育水稻(Oryza sativa L.)农垦58S(NK58S)的光合日变化和光抑制.06:00～09:00,NK58S的光抑制不明显,此时的光合功能下调以叶黄素循环为主;10:00～12:00,耗散比能流(DIo/RC)及光反应中心关闭净速率(dV/dto)增加,受体侧电子传递受阻(ψo下降),活性反应中心密度(Do)降低,NK58S光抑制加剧,PSⅡ反应中心发生失活.荧光暗弛豫分析与抑制剂处理结果表明,状态转换、叶黄素循环和PSⅡ反应中心失活均能有效保护NK58S免遭强光损伤.叶黄素循环相对于反应中心失活,前者是NK58S对强光胁迫的快速反应,在光强相对较弱时发挥主要作用,而后者在叶黄素循环达到饱和时对保护剩余活性反应中心起主要作用.     

Evolution and functional properties of photosystem II light harvesting complexes in eukaryotes

Photoautotrophic organisms, the major agent of inorganic carbon fixation into biomass, convert light energy into chemical energy. The first step of photosynthesis consists of the absorption of solar energy by pigments binding protein complexes named photosystems. Within photosystems, a family of proteins called Light Harvesting Complexes (LHC), responsible for light harvesting and energy transfer to reaction centers, has evolved along with eukaryotic organisms. Besides light absorption, these proteins catalyze photoprotective reactions which allowed functioning of oxygenic photosynthetic machinery in the increasingly oxidant environment. In this work we review current knowledge of LHC proteins serving Photosystem II. Balance between light harvesting and photoprotection is critical in Photosystem II, due to the lower quantum efficiency as compared to Photosystem I. In particular, we focus on the role of each antenna complex in light harvesting, energy transfer, scavenging of reactive oxygen species, chlorophyll triplet quenching and thermal dissipation of excess energy. This article is part of a Special Issue entitled: Photosystem II.     

Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: Catalyst identification,location and oligomeric structure

Photosynthetic conversion of light energy into chemical energy occurs in sheet-like membrane-bound compartments called thylakoids and is mediated by large integral membrane protein-pigment complexes called reaction centers (RCs). Oxygenic photosynthesis of higher plants, cyanobacteria and algae requires the symbiotic linking of two RCs, photosystem II (PSII) and photosystem I (PSI), to split water and assimilate carbon dioxide. Worldwide there is a large research investment in developing RC-based hybrids that utilize the highly evolved solar energy conversion capabilities of RCs to power catalytic reactions for solar fuel generation. Of particular interest is the solar-powered production of H₂, a clean and renewable energy source that can replace carbon-based fossil fuels and help provide for ever-increasing global energy demands. Recently, we developed thylakoid membrane hybrids with abiotic catalysts and demonstrated that photosynthetic Z-scheme electron flow from the light-driven water oxidation at PSII can drive H₂ production from PSI. One of these hybrid systems was created by self-assembling Pt-nanoparticles (PtNPs) with the stromal subunits of PSI that extend beyond the membrane plane in both spinach and cyanobacterial thylakoids. Using PtNPs as site-specific probe molecules, we report the electron microscopic (EM) imaging of oligomeric structure, location and organization of PSI in thylakoid membranes and provide the first direct visualization of photosynthetic Z-scheme solar water-splitting biohybrids for clean H₂ production.     

The currently accepted model of primary energy conversion in the plant photosystems must be substantially modernized

According to the current model of primary events in plants, electronic excitations generated in antenna chlorophylls (Chl) by the light rapidly migrate within vast Chl ensembles, reach the reaction centres (RCs) and initiate the primary photoreactions of electron transfer from the RC special pairs (P700 and P680 in plant photosystems). A minor portion of electronic excitations is lost en route, in particular, via fluorescence of Chl a. A number of fluorescence parameters in vivo had been reliably established in many independent studies. Based on these parameters, the author calculated a dimensionless value, the ratio of fluorescence yields emitted by PS-2 and PS-1 Chl a ensembles. The ratio proved to differ 5-10 times from that obtained in experiments. Such a divergence seems to indicate an internal discrepancy in the currently used model. The author proposes a substantial modernization of the model by introducing a new subpicosecond state for RCs, which precedes the primary reaction of electron transfer from the RC special pairs.     

Regulation of the primary photosynthesis processes

The mechanisms of primary processes of photosynthesis and macromolecular conformational changes that control the efficiency of primary energy transformation in photosynthesis are discussed. Special attention is focused on the analysis of chlorophyll fluorescence as an integrated parameter indicative of the efficiency and dynamics of primary steps of photosynthesis. Sharp changes in environmental conditions and other unfavorable factors may lead to the distortions of the coupling between consecutive electron transfer steps. As a result, an excess of electrons and/or electronic excitation energy may form at some sites of the electron transport chain. This may lead to the generation of reactive oxygen species responsible for the subsequent oxidative stress. The results of the application of these data in the areas of biotechnology and ecology are demonstrated.     

Charge Separation, Stabilization, and Protein Relaxation in Photosystem II Core Particles with Closed Reaction Center

The fluorescence kinetics of cyanobacterial photosystem II (PSII) core particles with closed reaction centers (RCs) were studied with picosecond resolution. The data are modeled in terms of electron transfer (ET) and associated protein conformational relaxation processes, resolving four different radical pair (RP) states. The target analyses reveal the importance of protein relaxation steps in the ET chain for the functioning of PSII. We also tested previously published data on cyanobacterial PSII with open RCs using models that involved protein relaxation steps as suggested by our data on closed RCs. The rationale for this reanalysis is that at least one short-lived component could not be described in the previous simpler models. This new analysis supports the involvement of a protein relaxation step for open RCs as well. In this model the rate of ET from reduced pheophytin to the primary quinone Q_A is determined to be 4.1 ns⁻¹. The rate of initial charge separation is slowed down substantially from ∼170 ns⁻¹ in PSII with open RCs to 56 ns⁻¹ upon reduction of Q_A. However, the free-energy drop of the first RP is not changed substantially between the two RC redox states. The currently assumed mechanistic model, assuming the same early RP intermediates in both states of RC, is inconsistent with the presented energetics of the RPs. Additionally, a comparison between PSII with closed RCs in isolated cores and in intact cells reveals slightly different relaxation kinetics, with a ∼3.7 ns component present only in isolated cores.     

Conformational regulation of charge recombination reactions in a photosynthetic bacterial reaction center

In bright light the photosynthetic reaction center (RC) of Rhodobacter sphaeroides stabilizes the P(+)(870).Q(-)(A) charge-separated state and thereby minimizes the potentially harmful effects of light saturation. Using X-ray diffraction we report a conformational change that occurs within the cytoplasmic domain of this RC in response to prolonged illumination with bright light. Our observations suggest a novel structural mechanism for the regulation of electron transfer reactions in photosynthesis.     

Trapped conformational states of semiquinone (D+*QB-*) formed by B-branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers

The reaction center (RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones QA and QB, involving a conformational gating step. In this work, conformational states of D+*QB-* were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D+*QB-* formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D+*QB-* in mutant RCs illuminated while freezing formed in approximately 95% of the sample did not decay (tau >107 s) at 80 K (also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active QB-*'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (approximately 70%) of QB, structurally located in the distal site, was not stably reducible, whereas the minority (approximately 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D+*QB-* states is attributed to the relaxation of protein residues and internal water molecules that stabilize D+*QB-*. These results demonstrate energetically significant conformational changes involved in stabilizing the D+*QB-* state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.     

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm²) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure—polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC?+?QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC?+?QD) complexes.

     

Preferential binding of equine ferricytochrome c to the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

Redox titration of horse heart cytochrome c (cyt c), in the presence of varying concentrations of detergent-solubilized photosynthetic reaction center (RC) from Rhodobacter sphaeroides, revealed an RC concentration-dependent decrease in the measured cyt c midpoint potential that is indicative of a 3.6 +/- 0.2-fold stronger binding affinity of oxidized cytochrome to a single binding site. This effect was correlated with preferential binding in the functional complex by redox titration of the fraction of RCs exhibiting microsecond, first-order, special pair reduction by cytochrome. A binding affinity ratio of 3.1 +/- 0.4 was determined by this second technique, confirming the result. Redox titration of flash-induced intracomplex electron transfer also showed the association in the electron transfer-active complex to be strong, with a dissociation constant of 0.17 +/- 0.03 microM. The tight binding is associated with a slow off-rate which, in the case of the oxidized form, can influence the kinetics of P(+) reduction. The pitfalls of the common use of xenon flashlamps to photoexcite fast electron-transfer reactions are discussed with relation to the first electron transfer from primary to secondary RC quinone acceptors. The results shed some light on the diversity of kinetic behavior reported for the cytochrome to RC electron-transfer reaction.