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.

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.

Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre.

The detergent effect on Cytochrome b559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1-D2-Cyt b559 complex preparations. Various n-dodecyl-beta-D-maltoside concentrations from 0 to 0.2% (w/v) were used to stabilise the D1-D2-Cyt b559 complexes. Low spin heme EPR spectra were obtained but the g(z) feature positions changed depending on the detergent conditions Redox potentiometric titrations showed a unique redox potential cytochrome b559 form (E'm = + 123-150 mV) in all the D1-D2-Cyt b559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome b559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.

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.

Unusual tolerance to high temperatures in a new herbicide-resistant D1 mutant from Glycine max (L.) Merr. cell cultures deficient in fatty acid desaturation.

The unusual tolerance to heat stress of STR7, an atrazine-resistant mutant isolated from photosynthetic cell-suspension cultures of soybean (Glycine max L. Merr. cv. Corsoy) and characterized previously [M. Alfonso et al. (1996) Plant Physiol 112:1499-1508] has been studied. The STR7 mutant maintained normal growth and fluorescence parameters at higher temperatures than the wild type (WT). The temperature for 50% inactivation of the oxygen-evolving activity of STR7 thylakoids was 13 degrees C higher than in the WT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with specific antibodies revealed that the integrity of photosystem II in the STR7 mutant was maintained at higher temperatures than in the WT. This unusual intrinsic tolerance to high temperatures contrasted with the higher sensitivity to heat stress reported as a feature linked to the triazine-resistance trait. The chloroplast membrane of STR7 accumulated an unusually high content of saturated C16:0 and reduced levels of C16:1 and C18:3 unsaturated fatty acids compared with the WT. Among all the lipid classes, chloroplastic lipids synthesized via the prokaryotic pathway (mono-galactosyl-diacyl-glycerol, phosphatidylglycerol and di-galactosyl-diacyl-glycerol), which represented more than 75% of the total lipid classes, showed the most substantial differences in C16:0 and C18:3 levels. In addition, changes in the physicochemical properties of the thylakoid membrane and chloroplast ultrastructure were also detected.

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.

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.

Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5-6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong-acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.

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.

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.

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.

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.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II.

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0-8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3- is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of approximately 2.5 microM and 20-34 microM when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20-34 microM is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

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.

Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB- states, and increased sensitivity to light stress.

We have isolated several herbicide-resistant cell lines from photosynthetic cell suspensions of soybean (Glycine max) that possessed different levels of herbicide resistance, photosystem II activity, and chlorophyll a/b ratio. We have further studied the STR7 mutant, which showed the highest level of resistance to atrazine as well as a cross-resistance to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (50- and 3-fold, respectively, compared with the wild type). Sequencing of the psbA gene (coding for the D1 polypeptide of photosystem II) from this mutant revealed a single change, serine-268 to proline, in the D1 protein. To our knowledge, this substitution has not previously been described in any photosynthetic organism. In addition to affecting atrazine resistance, this single amino acid change caused a decrease in the electron transfer rate between the secondary acceptors QA and QB and a stabilization of the S2QB- and S3QB- states. The mutant also showed a larger antenna size, an increase in non-QB-reducing centers, and a higher sensitivity to light stress. The unusual stability of the S2QB- and S3QB- states indicates that STR7 belongs to a new class of QB-site mutants.

Photoinhibition of photosystem II from higher plants. Effect of copper inhibition.

Strong illumination of Cu(II)-inhibited photosystem II membranes resulted in a faster loss of oxygen evolution activity compared with that of the intact samples. The phenomenon was oxygen- and temperature-dependent. However, D1 protein degradation rate was similar in both preparations and slower than that found in non-oxygen evolving PSII particles (i.e. Mn-depleted photosystem II). These results seem to indicate that during illumination Cu(II)-inhibited samples do not behave as a typical non-oxygen evolving photosystem II. Cytochrome b559 was functional in the presence of Cu(II). The effect of Cu(II) inhibition decreased the amount of photoreduced cytochrome b559 and slowed down the rate of its photoreduction. The presence of Cu(II) during illumination seems to protect P680 against photodamage as occurs in photosystem II reaction centers when the acceptor side is protected. The data were consistent with the finding that production of singlet oxygen was highly reduced in the preparations treated with Cu(II). EPR spin trapping experiments showed that inactivation of Cu(II)-treated samples was dominated by hydroxyl radical, and the loss of oxygen evolution activity was diminished by the presence of superoxide dismutase and catalase. These results indicate that the rapid loss of oxygen evolution activity in the presence of Cu(II) is mainly due to the formation of .OH radicals from superoxide ion via a Cu(II)-catalyzed Haber-Weiss mechanism. Considering that this inactivation process was oxygen-dependent, we propose that the formation of superoxide occurs in the acceptor side of photosystem II by interaction of molecular oxygen with reduced electron acceptor species, and thus, the primarily Cu(II)-inhibitory site in photosystem II is on the acceptor side.

Cu(II)-inhibitory effect on photosystem II from higher plants. A picosecond time-resolved fluorescence study.

The influence of Cu(II) inhibition on the primary reactions of photosystem II (PSII) electron transport was studied by picosecond time-resolved fluorescence on isolated PSII membranes. The fluorescence decay from Cu(II)-inhibited PSII centers showed a dominant amplitude of a fast phase (100-300 ps) similar to PSII centers in the uninhibited "open state" and minor contributions of components around 600 ps and 2.6 ns. These data indicate efficient primary charge separation in PSII membranes incubated with Cu(II). The quantum yield of primary reactions in the inhibited PSII centers was similar to that of "open" PSII centers. Kinetic analysis of the decay curves in the framework of the exciton/radical pair equilibrium model showed no significant changes in the rate constants associated with the charge separation/recombination equilibrium. However, in closed centers (QA reduced), a decrease in the rate constant K23, associated with the back-reaction of a relaxed radical pair, by a factor of 4 was calculated. The free energy losses upon primary charge separation (delta G1) and during subsequent radical pair relaxation (delta G2) were also determined in Cu(II)-inhibited centers and were compared with uninhibited centers. No changes in the delta G1 values and a significant decrease in the delta G2 values were found as compared with those of control PSII centers in the "closed" state. These data indicate that Cu(II) does not affect primary radical pair formation, but strongly affects the formation of a relaxed radical pair, by neutralizing the negative charge on QA- and eliminating the repulsive interaction between Pheo- and QA- and/or by modifying the general dielectric properties of the protein region, surrounding these cofactors. Moreover, a close attractive interaction between Pheo-, QA-, and Cu2+ can be proposed. Our results are in good agreement with very recent EPR results indicating an additional effect of Cu2+ on the acceptor side [Jegerschöld et al. (1995) Biochemistry 34, 12747-12758].

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes.

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800→B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800-B850 gap from ∼750 cm(\s-1) at 0.1 MPa to ∼900 cm(-1) at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800-850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800-B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C9-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (∼-0.4 cm(01)/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm(01)/MPa). The shift rate for B850 at 4.2 K is-0.28 cm(-1)/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.

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.

Detergent-induced reversible denaturation of the photosystem II reaction center: implications for pigment-protein interactions.

Incubation of the D1-D2-cytochrome b559 complex with Triton X-100 modified the protein secondary structure, caused significant spectral modifications, and reduced the formation of light-induced spin-polarized triplet electron paramagnetic resonance (EPR) signal. After 24 h of incubation, the absorption spectrum shifted from 675.5 to 671.5 nm and the fluorescence spectrum shifted from 682 to 672 nm. These shifts were accompanied by an increase in the chlorophyll fluorescence yield and by decreases in the intensity of the circular dichroism in the red region and the secondary electron transport activity. The intensity of the light-induced triplet EPR signal was also markedly reduced in the same experimental conditions. Substitution of dodecyl beta-maltoside for Triton X-100 reversed all the above-mentioned parameters to the values exhibited by the native D1-D2-Cyt b559 complex, including the characteristic triplet EPR signal. We concluded that all observed changes were due to the destruction of P680 with Triton X-100 and to the reestablishment of P680 in the presence of dodecyl beta-maltoside. The easier but certainly not the only possible explanation to all these phenomena is to consider a dimeric structure for P680, at least in its ground state, where interactions take place within the two dimeric chromophores and with the apoprotein. Such a dimeric structure would be very sensitive to small modifications of the P680 domain, which convert the dimer absorbing at 680 into two chlorophyll monomers absorbing near 670 nm. The dodecyl beta-maltoside reestablished the structure of the native P680 domain.