Reconstitution of photosynthetic reaction centers and core antenna-reaction center complexes in liposomes and their thermal stability

Photosynthetic reaction centers (RCs) and their core light-harvesting complexes (LH1-RCs), purified from a thermophile, Thermochromatium (T.) tepidum, and a mesophile, Allochromatium (A.) vinosum, were reconstituted into liposomes. The RC and the LH1-RC in the reconstituted liposomes were found intact from the absorption spectra at about 4 and 40 degrees C respectively. The thermal stability of the RCs of T. tepidum in the liposome was dependent on whether they were surrounded directly by lipids or by the core light-harvesting complexes. The results show that the RC of T. tepidum gains its thermostability through interactions with the LH1. These results are consistent with the result that the thermal stability of the LH1 in T. tepidum is similar in both the reconstituted LH1-RC liposome and ICM. This is clearly different from the mesophilic bacterium, A. vinosum. The thermal stability of RC was also affected by its subunit constitution: the RC containing a cytochrome subunit was more thermostable than the cytochrome-detached RC. This suggests that the cytochrome subunit might play a role in protecting the special pair pigments from denaturation. The thermal denaturation showed a second-order reaction dependence on time. The interaction of the pigments with proteins and/or lipids might be the cause of the second-order reaction profile.     

Resonance Raman studies of genetically modified reaction centers from Rhodobacter capsulatus

Resonance Raman (RR) spectra are reported for the photosynthetic reaction center (RC) proteins from Rhodobacter capsulatus wild type and the genetically modified systems GluL104----Leu and HisM200----Leu. The spectra were obtained with a variety of excitation wavelengths, spanning the UV, violet, and yellow-green regions of the absorption spectrum, and at temperatures of 30 and 200 K. The RR data indicate that the structures of the bacteriochlorin pigments in RCs from Rb. capsulatus wild type are similar to those in RCs from Rhodobacter sphaeroides wild type. The data also show that the amino acid modifications near the primary electron acceptor (GluL104----Leu) and special pair (HisM200----Leu) perturb only those bacteriochlorin pigments near the site of the mutation and do not influence the structures of the other pigments in the RC. In the case of the GluL104----Leu mutant, elimination of the hydrogen bond to the C9 keto group of BPhL results in frequency shifts of RR bands of certain skeletal modes of the macrocycle. This allows the assignment of bands to the individual BPhL and BPhM pigments. In the case of the HisM200----Leu mutant, in which the special pair is comprised of a bacteriochlorophyll (BChl)-bacteriopheophytin (BPh) heterodimer rather than the BChl2 unit bound in the wild type, certain skeletal vibrations due to the additional BPh unit are identified. The frequencies of these modes are similar to those of the analogous vibrations BPhL and BPhM, which indicates that the structure of the BPh in the heterodimer is not unusual in any discernible way.(ABSTRACT TRUNCATED AT 250 WORDS)     

去镁叶绿素置换细菌去镁叶绿素对紫细菌反应中心皮秒荧光的影响

选择597 nm作为激发波长,探测范围为600～900 nm的荧光特性,分析了天然反应中心和两种去镁叶绿素置换的紫细菌反应中心的荧光发射光谱.借助细菌叶绿素、细菌去镁叶绿素和植物去镁叶绿素的荧光光谱,对相关组分进行了归类.实验结果表明选择性地置换细菌去镁叶绿素影响了荧光光谱的组成.在天然反应中心、BpheB置换的反应中心和BpheA,B置换的反应中心中可分别解析到4、3和2个荧光发射组分.研究肯定荧光发射组分与去镁叶绿素的结合存在对应关系.实验还分别在686.4、674.1和681.1 nm处测定了不同反应中心内的原初电子供体P的激发态通过荧光衰减的过程,观测到衰减动力学上的差异.说明去镁叶绿素置换影响了细菌反应中心内激发光能传递和原初光化学反应过程.     

Replacement of bacteriopheophytin in reaction centers from Rhodobacter sphaeroides RS601 with plant pheophytin

In the presence of acetone and an excess of exogenous plant pheophytins,bacteriopheophytins in the reaction centers from Rhodobacter sphaeroides RS601 were replaced by pheophytins at sites HA and HB,when incubated at 43.5℃ for more than 15 min.The substitution of bacteriopheophytins in the reaction centers was 50% and 71% with incubation of 15 and 60 min,respectively.In the absorption spectra of pheophytin-replaced reaction centers (Phe RCs),bands assigned to the transition moments QX (537 nm) and QY (758 nm) of bacteriopheophytin disappeared,and three distinct bands assigned to the transition moments QX (509/542 nm) and QY (674 nm) of pheophytin appeared instead.Compared to that of the control reaction centers,the photochemical activities of Phe RCs are 78% and 71% of control,with the incubation time of 15 and 60 min.Differences might exist between the redox properties of Phe RC and of native reaction centers,but the substitution is significant,and the new system is available for further studies.     

Circular symmetry of the light-harvesting 1 complex from Rhodospirillum rubrum is not perturbed by interaction with the reaction center

The effect of the interaction of the reaction center (RC) upon the geometrical arrangement of the bacteriochlorophyll a (BChla) pigments in the light-harvesting 1 complex (LH1) from Rhodospirillum rubrum has been examined using single molecule spectroscopy. Fluorescence excitation spectra at 1.8 K obtained from single detergent-solubilized as well as single membrane-reconstituted LH1-RC complexes showed predominantly (>70%) a single broad absorption maximum at 880-900 nm corresponding to the Q(y) transition of the LH1 complex. This absorption band was independent of the polarization direction of the excitation light. The remaining complexes showed two mutually orthogonal absorption bands in the same wavelength region with moderate splittings in the range of DeltaE = 30-85 cm(-1). Our observations are in agreement with simulated spectra of an array of 32 strongly coupled BChla dipoles arranged in perfect circular symmetry possessing only a diagonal disorder of 相似文献   

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Effect of dipyridamole on the recombination kinetics between photooxidized bacteriochlorophyll and photoreduced primary quinone in reaction centres of purple bacteria

The action of dipyridamole (DIP) on dark recombination between the photooxidized special pair bacteriochlorophyll BChl2+ and reduced primary quinone acceptor Q(A)- in the reaction centres (RCs) of the bacteria Rhodobacter sphaeroides was studied in the presence of different detergents (LDAO, Triton X-100, sodium cholate, sodium dodecyl sulfate). DIP accelerated this reaction approximately 4-5-fold. In RCs with the extracted H-subunit, the effect of DIP was observed at lower concentrations. The possibility of modification of the RC structure-dynamic state by DIP (including changes in RC hydrogen bonds) is proposed. The modification obviously disturbs the processes of the long-life electrostatic stabilization of Q(A)-.     

Reconstitution of Photosystem Ⅱ Reaction Center with Cu-ChlorophyⅡ a

An Isolated photosystem （PS） II reaction center （RC） with altered pigment content was obtained by chemical exchange of native chlorophyll a （Chl） with externally added Cu-Chl a （Cu-Chl）. Pigment composition and spectroscopic properties of the RC exchanged with Cu-Chl were compared with native RC and RC treated with Chl In the same way. High-performance liquid chromatography analysis showed approximately 0.5 Cu-Chl per two pheophytln in the Cu-Chl-reconstltuted RC preparation. Insertion of Cu-Chl resulted in a decrease In absorption at 670 nm and an Increase at 660 nm, suggesting that the peripheral Chl may have been displaced. Fluorescence emission spectra of the Cu-Chl-reconstituted RC displayed a marked decrease In fluorescence yield and a blue shift of the band maximum, accompanied by the appearance of a broad peak at a shorter wavelength, Indicating that energy transfer In the modified RC was disturbed by Cu-Chl, a quencher of the excited state. However, there were few differences in the circular dichrolsm （CD） spectra, suggesting that the arrangement of pigments and proteins responsible for the CD signal was not significantly affected. In addition, no obvious change In peptlde components was found after the exchange procedure.     

PMS: Photosystem I electron donor or fluorescence quencher

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.     

Replacement of bacteriopheophytin in reaction centers fromRhodobacter sphaeroides RS601 with plant pheophytin

In the presence of acetone and an excess of exogenous plant pheophytins, bacterio-pheophytins in the reaction centers from Rhodobacter sphaeroides RS601 were replaced by pheophytins at sites HA and HB, when incubated at 43.5℃ for more than 15 min. The substitution of bacteriopheophytins in the reaction centers was 50% and 71% with incubation of 15 and 60 min, respectively. In the absorption spectra of pheophytin-replaced reaction centers (Phe RCs), bands assigned to the transition moments Qx (537 nm) and QY (758 nm) of bacteriopheophytin disappeared, and three distinct bands assigned to the transition moments Qx (509/542 nm) and QY (674 nm) of pheophytin appeared instead. Compared to that of the control reaction centers, the photochemical activities of Phe RCs are 78% and 71% of control, with the incubation time of 15 and 60 min. Differences might exist between the redox properties of Phe RC and of native reaction centers, but the substitution is significant, and the new system is available for further     

Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+

Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.     

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.     

Selective perturbation of the second electron transfer step in mutant bacterial reaction centers

In order to specifically perturb the primary electron acceptor B(A) -- a monomeric bacteriochlorophyll (BChl) a -- involved in bacterial photosynthetic charge separation (CS), the protein environment of B(A) in the reaction center (RC) of Rhodobacter sphaeroides was modified by site-directed mutagenesis. Isolated RCs were characterized by redox titrations, low temperature optical spectroscopy, ENDOR/TRIPLE resonance spectroscopy and femtosecond time-resolved spectroscopy. Two mutations were studied: In the GS(M203) mutant a serine is introduced near the ring E keto group of B(A), while in FY(L146) a phenylalanine near the ring A acetyl group of B(A) is replaced by tyrosine. In all mutations the oxidation potential of the primary electron donor P as well as the electronic structure of both the P(*+) radical cation and the radical anion of the secondary electron acceptor, H(A)(*-), are not significantly altered compared to the wild type (WT), while changes of the optical absorption spectra at 77 K in the BChl Q(X) and Q(Y) regions are observed. The GS(M203) mutation only leads to a minor retardation of the CS reactions at room temperature, whereas for FY(L146) significant deviations from the native electron transfer (ET) rates could be detected: In addition to a faster first (2.9 ps) and a slower second (1 ps) ET step, a new 8-ps time constant was found in the FY(L146) mutant, which can be ascribed to a fraction of RCs with slowed down secondary ET. The results allow us to address the functional role of the acetyl group of B(A) and question the role of the free energy changes as the main determining factor of ET rates in RCs. It is concluded that structural rearrangements alter the electronic coupling between the pigments and thereby influence the rate of fast CS.     

Temperature dependent UV-Vis spectral changes in hydrogen-and deuterium-bonded photosynthetic reaction centers of Rhodobacter sphaeroides

The UV-Vis absorption spectra of detergent-isolated hydrogen-and deuterium-bonded reaction centers (RCs) from Rhodobacter sphaeroides PUC 705Ba were examined as a function of temperature between 20 and 55 °C. The enthalpy and entropy of denaturation for the specimens was determined, revealing that their process of thermal denaturation is significantly different. Deuterium-bonded RCs are most stable at 37 °C, rather than at room temperature, and undergo a “cold denaturation” as the temperature is lowered to room temperature. At room temperature the addition of 1,3,5-heptanetriol brought the deuterium-bonded RC back to its more stable configuration. Hence the hydrogen bonding interactions in the RC do influence its conformation and this is reflected in the microenvironment of its associated pigments.     

The effect of an applied electric field on the charge recombination kinetics in reaction centers reconstituted in planar lipid bilayers.

Reaction Centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides were incorporated in planar bilayers made from monolayers derived from liposomes reconstituted with purified RCs. The photocurrents associated with the charge recombination process between the reduced primary quinone (QA-) and the oxidized bacteriochlorophyll donor (D+) were measured as a function of voltage (-150 mV less than V less than 150 mV) applied across the bilayer. When QA was the native ubiquinone (UQ) the charge recombination was voltage independent. However, when UQ was replaced by anthraquinone (AQ), the recombination time depended on the applied voltage V according to the relation tau = 8.5 X 10(-3) eV/0.175S. These results were explained by a simple model in which the charge recombination from UQ- proceeds directly to D+ while that from AQ occurs via a thermally activated intermediate state, D+I-QA, where I is the intermediate acceptor. The voltage dependence arises from an electric field induced change in the energy gap, delta G0, between the states D+I-QA and D+IQA-. This model is supported by the measured temperature dependence of the charge recombination time, which for RCs with AQ gave a value of delta G0 = 340 +/- 20 meV. In contrast, delta G0 for RCs with UQ as the primary acceptor, is sufficiently large (approximately 550 meV) so that even in the presence of the field, the direct pathway dominates. The voltage dependence shows that the electron transfer from I- to QA is electrogenic. From a quantitative analysis of the voltage dependence on the recombination rate it was concluded that the component of the distance between I and QA along the normal to the membrane is about one-seventh of the thickness of the membrane. This implies that the electron transfer from I to Q contributes at least one-seventh to the potential generated by the charge separation between D+ and QA-.     

Reconstitution of the type-1 active site of the H145G/A variants of nitrite reductase by ligand insertion

Variants of the copper-containing nitrite reductase (NiR) of Alcaligenes faecalis S6 were constructed by site-directed mutagenesis, by which the C-terminal histidine ligand (His145) of the Cu in the type-1 site was replaced by an alanine or a glycine. The type-1 sites in the NiR variants as isolated, are in the reduced form, but can be oxidized in the presence of external ligands, like (substituted) imidazoles and chloride. The reduction potential of the type-1 site of NiR-H145A reconstituted with imidazole amounts to 505 mV vs NHE (20 degrees C, pH 7, 10 mM imidazole), while for the native type-1 site it amounts to 260 mV. XRD data on crystals of the reduced and oxidized NiR-H145A variant show that in the reduced type-1 site the metal is 3-coordinated, but in the oxidized form takes up a ligand from the solution. With the fourth (exogenous) ligand in place the type-1 site is able to accept electrons at about the same rate as the wt NiR, but it is unable to pass the electron onto the type-2 site, leading to loss of enzymatic activity. It is argued that the uptake of an electron by the mutated type-1 site is accompanied by a loss of the exogenous ligand and a concomitant rise of the redox potential. This rise effectively traps the electron in the type-1 site.     

Isolation and characterization of homodimeric type-I reaction center complex from Candidatus Chloracidobacterium thermophilum, an aerobic chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ～483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.     

X-Ray structure determination of three mutants of the bacterial photosynthetic reaction centers from Rb. sphaeroides; altered proton transfer pathways

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the reduction of a bound quinone molecule Q(B) is coupled with proton uptake. When Asp-L213 is replaced by Asn, proton transfer is inhibited. Proton transfer was restored by two second-site revertant mutations, Arg-M233-->Cys and Arg-H177-->His. Kinetic effects of Cd(2+) on proton transfer showed that the entry point in revertant RCs to be the same as in the native RC. The structures of the parental and two revertant RCs were determined at resolutions of 2.10, 1.80, and 2.75 A. From the structures, we were able to delineate alternate proton transfer pathways in the revertants. The main changes occur near Glu-H173, which allow it to substitute for the missing Asp-L213. The electrostatic changes near Glu-H173 cause it to be a good proton donor and acceptor, and the structural changes create a cavity which accommodates water molecules that connect Glu-H173 to other proton transfer components.     

The redox midpoint potential of the primary quinone of reaction centers in chromatophores of Rhodobacter sphaeroides is pH independent

The redox midpoint potential (E (m)) of the primary quinone of bacterial reaction centers, Q(A), in native membranes (chromatophores) measured by redox potentiometry is reported to be pH dependent (-60 mV/pH) up to a highly distinctive pK ( a ) (9.8 in Rba. sphaeroides) for the reduced state. In contrast, the E (m) of Q(A) in isolated RCs of Rba. sphaeroides, although more variable, has been found to be essentially pH-independent by both redox potentiometry and by delayed fluorescence, which determines the free energy (DeltaG (P*A)) of the P(+)Q (A) (-) state relative to P*. Delayed fluorescence was used here to determine the free energy of P(+)Q (A) (-) in chromatophores. The emission intensity in chromatophores is two orders of magnitude greater than from isolated RCs largely due to the entropic effect of antenna pigments "drawing out" the excitation from the RC. The pH dependence of DeltaG (P*A) was almost identical to that of isolated RCs, in stark contrast with potentiometric redox titrations of Q(A). We considered that Q(A) might be reduced by disproportionation with QH(2) through the Q(B) site, so the titration actually reflects the quinone pool, giving the -60 mV/pH unit dependence expected for the Q/QH(2) couple. However, the parameters necessary to achieve a strong pH-dependence are not in good agreement with expected properties of Q(A) and Q(B). We also consider the possibility that the time scale of potentiometric titrations allows the reduced state (Q (A) (-) ) to relax to a different conformation that is accompanied by stoichiometric H(+) binding. Finally, we discuss the choice of parameters necessary for determining the free energy level of P(+)Q (A) (-) from delayed fluorescence emission from chromatophores of Rba. sphaeroides.