The photoreaction center of Rhodospirillum rubrum mutant strain F24.1.

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665-670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7-8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7-8% of typical photoreaction center.

Spin label electron paramagnetic resonance study in thylakoid membranes from a new herbicide-resistant D1 mutant from soybean cell cultures deficient in fatty acid desaturation.

The effect of fatty acid desaturation on lipid fluidity in thylakoid membranes isolated from the STR7 mutant was investigated by electron paramagnetic resonance (EPR) using spin label probes. The spectra of both 5- and 16-n-doxylstearic acid probes were measured as a function of the temperature between 10 and 305 K and compared to those of the wild type. This complete thermal evolution provides a wider picture of the dynamics. The spectra of the 5-n-doxylstearic acid probe as well as their temperature evolution were identical in both STR7 mutant and wild type thylakoids. However, differences were found with the 16-n-doxylstearic acid probe at temperatures between 230 and 305 K. The differences in the thermal evolution of the EPR spectra can be interpreted as a 5-10 K shift toward higher temperatures of the probe motional rates in the STR7 mutant as compared with that in the wild type. At temperatures below 230 K no differences were observed. The results indicated that the lipid motion in the outermost region of the thylakoids is the same in the STR7 mutant as in the wild type while the fluidity in the inner region of the STR7 mutant membrane decreases. Our data point out a picture of the STR7 thylakoid membrane in which the lipid motion is slower most probably as a consequence of fatty acid desaturation deficiency.

Pigment stoichiometry of a newly isolated D1-D2-Cyt b559 complex from the higher plant Beta vulgaris L.

Two D1-D2-Cyt b559 complexes with different pigment stoichiometry were isolated from the higher plant B. vulgaris. The procedures for isolating both complexes only differed in the washing time of the DEAE column with 50 mM Tris-HCl, pH 7.2, 0.05% Triton X-100 and 30 mM NaCl. When the column was washed until the eluate had an absorbance of 0.01 at 670 nm, the isolated D1-D2-Cyt b559 complex presented a pigment stoichiometry of 6 chlorophyll a, 2 beta-carotene, and 1 cytochrome b559 per 2 pheophytin a. In contrast, when the column was exhaustively washed until the eluate reached an absorbance of 0.005 at 670 nm, the complex had a stoichiometry of 4 chlorophyll a, 1 beta-carotene, and 1 cytochrome b559 per 2 pheophytin a. We think that the former stoichiometry corresponds to that of the native D1-D2-Cyt b559 complex. Moreover, both preparations showed 2 mol of pheophytin a per 1 mol of reaction center protein.

Effect of bicarbonate on the S2 multiline EPR signal of the oxygen-evolving complex in photosystem II membrane fragments.

Removal of bicarbonate from spinach photosystem II BBY particles by means of washing in a CO2-free medium results in the loss of their capability to accumulate the S2 multiline EPR signal upon continuous illumination at 190 K. Addition of 1 mM NaHCO3 before illumination leads to a 50-60% restoration of the multiline signal. Similarly, in BBY particles depleted of Mn by treatment with 1 M Tris-HCl (pH 8.0) and 0.5 M MgCl2, re-addition of MnCl2 in the presence of 1 mM NaHCO3 results in a partial restoration (approximately 30%) of the S2 multiline EPR signal of the Mn cluster, while in the absence of NaHCO3 no restoration is observed. The results provide further evidence that bicarbonate is essential for maintaining the Mn-containing oxygen-evolving complex of PS II in a functionally active form.

Light-induced absorption spectra of the D1-D2-cytochrome b 559 complex of Photosystem II: Effect of methyl viologen concentration.

The light-induced difference absorption spectra associated to the photo-accumulation of reduced pheophytin a were studied in the isolated D1-D2-Cyt b559 complex in the presence of variable methyl viologen concentrations and different illumination conditions under anaerobiosis. Depending on the methyl viologen/reaction centre ratio, the relative intensities of the spectral bands at 681.5+/-0.5, 667.0+/-0.5 and 542.5+/-0.5 nm were modified. The reduced pheophytin a located at the D1-branch of the complex absorbs at 681.7+/-0.5 nm, and at least two additional pigment species contribute to the Q(y) band of the difference absorption spectra with maxima at 667.0+/-0.5 and 680.5+/-0.5 nm. We propose the additional species correspond to a peripheral chlorophyll a and the pheophytin a located at the D2-branch of the complex, respectively. The blue absorbing chlorophyll at 667 nm is susceptible to chemical redox changes with a midpoint reduction potential of +470 mV. The Q(x) absorption bands of both pheophytins localised at the D2- and D1-branch of the D1-D2-Cyt b559 complex were at 540.7+/-0.5 and 542.9+/-0.5, respectively. The results indicated that the two pheophytin molecules can be photoreduced in the D1-D2-Cyt b559 complex in certain experimental conditions.

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c' and cytochrome c, are detected. A high potential iron-sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron-sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center.

Photobleaching of photosynthetic pigments in spinach thylakoid membranes. Effect of temperature, oxygen and DCMU.

The time dependence of photobleaching of photosynthetic pigments under high light illumination of isolated spinach thylakoid membranes at 22 and 4 degrees C was investigated. At 22 degrees C, the bleaching at 678, 472 and 436 nm was prominent but lowering the temperature up to 4 degrees C during illumination prevented the pigments from bleaching almost completely. The accelerating effect on pigment photobleaching by the presence of 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea)-(DCMU), a well-known inhibitor of the electron transport and known to prevent photosystem I (PSI) and photosystem II (PSII) against photoinhibitory damage, was also suppressed at low temperature. At 22 degrees C in the presence and absence of DCMU, the decrease of the absorption at 678 and 472 nm was accompanied by a shift to the shorter wavelengths. To check the involvement of reactive oxygen species in the process, pigment photobleaching was followed in anaerobiosis. The effects of the three different environmental factors--light, temperature and DCMU--on the dynamics of photobleaching are discussed in terms of different susceptibility of the main pigment-protein complexes to photoinhibition.

Resonance Raman and surface-enhanced resonance Raman spectra of LH2 antenna complex from Rhodobacter sphaeroides and Ectothiorhodospira sp. excited in the Qx and Qy transitions.

Well-resolved vibrational spectra of LH2 complex isolated from two photosynthetic bacteria, Rhodobacter sphaeroides and Ectothiorhodospira sp., were obtained using surface-enhanced resonance Raman scattering (SERRS) exciting into the Qx and the Qy transitions of bacteriochlorophyll a. High-quality SERRS spectra in the Qy region were accessible because the strong fluorescence background was quenched near the roughened Ag surface. A comparison of the spectra obtained with 590 nm and 752 nm excitation in the mid- and low-frequency regions revealed spectral differences between the two LH2 complexes as well as between the LH2 complexes and isolated bacteriochlorophyll a. Because peripheral modes of pigments contribute mainly to the low-frequency spectral region, frequencies and intensities of many vibrational bands in this region are affected by interactions with the protein. The results demonstrate that the microenvironment surrounding the pigments within the two LH2 complexes is somewhat different, despite the fact that the complexes exhibit similar electronic absorption spectra. These differences are most probably due to specific pigment-pigment and pigment-protein interactions within the LH2 complexes, and the approach might be useful for addressing subtle static and dynamic structural variances between pigment-protein complexes from different sources or in complexes altered chemically or genetically.

Langmuir-Blodgett and X-ray diffraction studies of isolated photosystem II reaction centers in monolayers and multilayers: physical dimensions of the complex.

The photosystem II (PSII) reaction center (RC) is a hydrophobic intrinsic protein complex that drives the water-oxidation process of photosynthesis. Unlike the bacterial RC complex, an X-ray crystal structure of the PSII RC is not available. In order to determine the physical dimensions of the isolated PSII RC complex, we applied Langmuir techniques to determine the cross-sectional area of an isolated RC in a condensed monolayer film. Low-angle X-ray diffraction results obtained by examining Langmuir-Blodgett multilayer films of alternating PSII RC/Cd stearate monolayers were used to determine the length (or height; z-direction, perpendicular to the plane of the original membrane) of the complex. The values obtained for a PSII RC monomer were 26 nm2 and 4.8 nm, respectively, and the structural integrity of the RC in the multilayer film was confirmed by several approaches. Assuming a cylindrical-type RC structure, the above dimensions lead to a predicted volume of about 125 nm3. This value is very close to the expected volume of 118 nm3, calculated from the known molecular weight and partial specific volume of the PSII RC proteins. This same type of comparison was also made with the Rhodobacter sphaeroides RC based on published data, and we conclude that the PSII RC is much shorter in length and has a more regular solid geometric structure than the bacterial RC. Furthermore, the above dimensions of the PSII RC and those of PSII core (RC plus proximal antenna) proteins protruding outside the plane of the PSII membrane into the lumenal space as imaged by scanning tunneling microscopy (Seibert, Aust. J. Pl. Physiol. 22, 161-166, 1995) fit easily into the known dimensions of the PSII core complex visualized by others as electron-density projection maps. From this we conclude that the in situ PSII core complex is a dimeric structure containing two copies of the PSII RC.

A study on the heterogeneity of the light-harvesting complex II from Ectothiorhodospira sp. after acid/chaotropic treatment.

The light-harvesting complex II of the purple bacteria has two strong near infrared electronic absorption bands, around 800 (B800) and 850 (B850) nm, arising from the Qy transitions of bacteriochlorophyll a. It was previously reported that under some specific acid/chaotropic conditions the B850 bacteriochlorophylls of the light-harvesting complex II of Ectothiorhodospira sp. are strongly reorganised. Part of these pigments absorbs at 843 nm while another set absorbs around 858 nm. The current work should investigate whether a mix of two different complexes could generate the 843- and 858-nm bands. Acid/chaotropic conditions inducing the reorganisation of B850 were reproduced on a sample bound to an ionic-exchange column. The chromatographic pattern was found strongly homogeneous. The findings indicate that the heterogeneity of the reorganised B850 results from two forms of differently structured bacteriochlorophylls bound to the same polypeptide backbone.

Site energies of active and inactive pheophytins in the reaction center of Photosystem II from Chlamydomonas reinhardtii.

It is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction center (RC) preparations is pheophytin a (Pheo a) within the D1 protein (Pheo(D1)), while Pheo(D2) (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the past two decades placed the Q(y)-states of Pheo(D1) and Pheo(D2) bands near 678-684 and 668-672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986 - 998; Cox et al. J. Phys. Chem. B 2009, 113, 12364 - 12374] of the electronic structure of the PSII RC reversed the assignment of the active and inactive Pheos, suggesting that the mean site energy of Pheo(D1) is near 672 nm, whereas Pheo(D2) (~677.5 nm) and Chl(D1) (~680 nm) have the lowest energies (i.e., the Pheo(D2)-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Q(y) absorption maxima at 676-680 nm [Germano et al. Biochemistry 2001, 40, 11472 - 11482; Germano et al. Biophys. J. 2004, 86, 1664 - 1672]. To provide more insight into the site energies of both Pheo(D1) and Pheo(D2) (including the corresponding Q(x) transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch Pheo(D1) is genetically replaced with chlorophyll a (Chl a). We show that the Q(x)-/Q(y)-region site energies of Pheo(D1) and Pheo(D2) are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803 - 8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.

Supramolecular arrangement of Rhodospirillum rubrum B880 holochrome as studied by radiation inactivation and electron paramagnetic resonance.

Oxidation of the B880 antenna holochrome gives rise to a 3.8-G linewidth electron paramagnetic resonance (EPR) signal that is considerably narrower than the 13-G signal of monomeric bacteriochlorophyll (Bchl) cation. Radiation inactivation was used to verify a model according to which this linewidth narrowing is due to delocalization over several Bchl molecules. Chromatophores of the photoreaction centerless mutant F24 of Rhodospirillum rubrum were subjected to different doses of gamma-radiation. This induced not only a decay of the EPR signal amplitude but also its linewidth broadening. According to target theory, the induced amplitude decay of the EPR signal had a target size of 10.5 kDa. This is attributed to an elementary structure (alpha1beta1Bchl2), whose number in the membrane would limit the rate of encounter with ferricyanide and thus the formation of unpaired spins. We applied Bernoulli statistics to predict, for a given survival probability of the signal, the number of surviving elementary structures in aggregates of (alpha1beta1Bchl2)n where n was varied from 4 to 7. Using an equation that predicted the Bchl special pair in the photo-reaction center, we were able to simulate the observed relationship between the EPR linewidth and the dose of radiation. The best fit was obtained with a hexameric structure alpha1beta1Bchl2)6.

Alkaline denaturation of the light-harvesting complex II from the purple bacterium Ectothiorhodospira sp.: kinetic evidence of the existence of the 780 nm upper exciton component of the B850 bacteriochlorophylls.

The light-harvesting complex II of the purple bacteria has two strong near-infrared electronic absorption bands around 800 (B800) and 850 (B850) nm, arising from the Q(y)() transitions of the bacteriochlorophyll a. In the present work, high concentrations of NaOH were used to study the destabilization of the complex of the Ectothiorhodospira sp. The majority of the bacteriochlorophylls were monomerized within 90 min of treatment. However, the kinetic patterns of the two near-infrared bands were remarkably different. After an instantaneous blue shift from 853 to 828 nm, B850 showed a first-order monomerization with a rate constant of -0.016 min(-1). This instantaneous blue shift was previously attributed to the deprotonation of a lysine and was independent of the monomerization process. The observed native B800 is in fact composed of two bands, one at 796 nm and the other at 780 nm. The band absorbing at 780 nm red shifted also instantaneously to 786-788 nm and then disappeared in a first-order process as B850. The other band absorbing at 796 nm has a two-step process of monomerization; after a rapid conversion a slower first-order process occurred with a rate constant of -0.025 min(-1). The similarity between the kinetic behaviors of B850 and the 780 nm band indicated a strong relationship between these two bands. Our interpretation of the results considers the 780 nm band as the upper exciton component of the B850 bacteriochlorophylls.

Phototrapping of doubly reduced monomeric bacteriochlorophyll in the photoreaction center of Ectothiorhodospira sp.

The photoreaction center from the purple sulfur bacterium Ectothiorhodospira sp. was illuminated in the presence of reduced cytochrome c or dithionite under anaerobic conditions. This treatment first caused the monoelectronic reduction of both molecules of bacteriopheophytin (Bph), phi A and phi B, as witnessed by the appearance of EPR and optical signals typical of singly-reduced bacteriochlorins. Continued illumination under the same reducing conditions caused both of these signals to disappear. Such disappearance was accompanied by a complete bleaching of the Qx and Qy absorption bands of Bph but not of the corresponding transitions of bacteriochlorophyll (Bchl). These phenomena are interpreted by a double reduction of phi A and phi B. As long as the medium remained reducing and anaerobic, these changes were stable. Prolonged illumination under the same reducing conditions finally led to the bleaching of the Qx (600 nm) and Qy (800 nm) bands of Bchl but not of the 880-nm band. This generated no EPR or 645-nm absorption signals due to singly-reduced Bchl. The bleaching kinetics of the 800-nm band was biphasic and paralleled a shift of the peak wavelength. This is interpreted by a double reduction of both molecules of monomeric Bchl BA and BB in an undetermined order. After bleaching of the 800-nm band has reached saturation, the absorbance ratio of the 800/880-nm absorption bands remains constant, as would be expected if the ultimate spectrum was that of the primary electron donor. These experiments demonstrate the photoreduction of Bchl and allow the absorption spectrum of the primary donor to be measured for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectral changes of the B800-850 antenna complex from Ectothiorhodospira sp. induced by detergent and salt treatment.

We have studied the effects of the detergent lauryl dimethylamine N-oxide and NaCl in the near infrared absorption spectra of the B800-850 antenna complex from Ectothiorhodospira sp. Strong spectral changes were induced on the BChl850 band by the lauryl dimethylamine N-oxide consisting of a blue shift, from 857 to 839-837 nm, and a hypochromism. No significant effects were detected on the BChl800 band in the same conditions. The changes were reversible after removing most of the detergent from the sample. Depending upon the detergent concentration in the solution, NaCl was also able to reverse the blueshift and increase the intensity of the 850 nm band close to the native values. Moreover, we have been able to separate both phenomena. Addition of 0.350 M NaCl after sample incubation with 0.15% (v/v) lauryl dimethylamine N-oxide for 30 min allowed a 9-10 nm redshift with no significant hyperchromism of the lowest energy band. We explained the overall effect of the detergent assuming that the lauryl dimethylamine N-oxide bound to the hydrophobic moiety of the complex and caused some protein conformational changes which affected the BChl850 domain without affecting that of the BChl800. The NaCl was able to circumvent these effects, most probably by acting directly on the BChl850 molecules or on the protein structure surrounding them.

The inhibitory mechanism of Cu(II) on the Photosystem II electron transport from higher plants.

In a previous paper, we reported that Cu(II) inhibited the photosynthetic electron transfer at the level of the pheophytin-QA-Fe domain of the Photosystem II reaction center. In this paper we characterize the underlying mechanism of Cu(II) inhibition. Cu(II)-inhibition effect was more sensitive with high pH values. Double-reciprocal plot of the inhibition of oxygen evolution by Cu(II) is shown and its corresponding inhibition constant, Ki, was calculated. Inhibition by Cu(II) was non-competitive with respect to 2,6-dichlorobenzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea and competitive with respect to protons. The non-competitive inhibition indicates that the Cu(II)-binding site is different from that of the 2,6-dichlorobenzoquinone electron acceptor and 3-(3,4-dichlorophenyl)-1,1-dimethylurea sites, the QB niche. On the other hand, the competitive inhibition with respect to protons may indicate that Cu(II) interacts with an essential amino acid group(s) that can be protonated or deprotonated in the inhibitory-binding site.

Oxido-reduction of B800-850 and B880 holochromes isolated from three species of photosynthetic bacteria as studied by electron-paramagnetic resonance and optical spectroscopy.

Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.

Stabilization of Isolated Photosystem II Reaction Center Complex in the Dark and in the Light Using Polyethylene Glycol and an Oxygen-Scrubbing System.

The photosystem II reaction center as isolated (O Nanba, K Satoh [1987] Proc Natl Acad Sci USA 84: 109-112) is quite dilute and very unstable. Precipitating the complex with polyethylene glycol and resuspending it in buffer without detergent concentrates the reaction center and greatly improves its stability at 4 degrees C in the dark as judged by light-induced electron transport activity. Furthermore, a procedure was developed to minimize photodestruction of polyethylene-glycol-concentrated material at room temperature in the light. The ability to stabilize the photosystem II reaction center should facilitate future photophysical, biochemical, and structural studies of the complex.

Pigment content of D1-D2-cytochrome b559 reaction center preparations after removal of CP47 contamination: an immunological study.

Isolated D1-D2-cytochrome b559 photosystem II reaction center preparations with pigment stoichiometry higher than 4 chlorophylls per 2 pheophytins can be contaminated with CP47 proximal antenna complex. Reaction center prepared by a modification of the Nanba-Satoh procedure and containing about 6 chlorophylls per 2 pheophytins showed immuno-cross-reactivity when probed with a monoclonal antibody raised against the CP47 polypeptide. Furthermore, they could be fractionated successfully by Superose-12 sieve chromatography into two different populations. The first few fractions off the column contained a more definitive 435 nm shoulder corresponding to increased chlorophyll content, and showed strong immuno-cross-reactivity with the CP47 antibody. The peak fractions off the column displayed a less prominent 435 nm shoulder, and did not cross-react with the antibody. Moreover, when a 6-chlorophyll preparation was mixed with Sepharose beads coupled to CP47 antibody, the eluted material corresponded to a preparation of about 4 chlorophylls per 2 pheophytins and did not show any cross-reaction with the antibody against CP47. The amount of CP47 protein in the 6-chlorophyll preparation as quantitated using Coomassie Blue staining or from gel blots was sufficient to account for most of the extra 2 chlorophylls. We conclude that D1-D2-cytochrome b559 preparations containing more than 4 chlorophylls per 2 pheophytins can be contaminated with small amounts of CP47-D1-D2-Cyt b559 complex and that native photosystem II reaction centers contain 4 core chlorophylls per 2 pheophytins.