Absorbance and fluorescence properties of the bacteriochlorophyll a reaction center complex and bacteriochlorophyll a protein in green bacteria

Absorbance, emission and excitation spectra were measured at both room and liquid-nitrogen temperatures for a photochemically active bacteriochlorophyll a reaction center complex and a bacteriochlorophyll a protein isolated from Chlorobium limicola and Chlorobium thiosulfatophilum. The low-temperature absorbance spectrum for the complex has a band centered at 833 nm, which is not seen in the spectrum of the bacteriochlorophyll a protein. We attribute this difference to a modification of the bacteriochlorophyll a protein in the active complex. The room-temperature fluorescence spectra for the bacteriochlorophyll a protein and the complex are similar, as are those measured at low temperatures. The 833-nm component of the low-temperature absorbance spectrum of the complex is relatively nonfluorescent.     

Bacteriopheophytin c in reaction center complexes of green photosynthetic bacteria

Two reaction center complexes prepared from cytoplasmic membranes of Chlorobium limicola f. thiosulfato-philum were compared by absorption and CD spectrophotometry. Bacteriopheophytin c (670 nm), which is optically active in one complex but not in the other, may serve as a secondary electron acceptor in the reaction center.     

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [N?veke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.     

Exciton interactions in dimers of bacteriochlorophyll and related molecules

Formalisms are developed for calculating the absorption wavelengths, dipole strengths and rotational strengths for dimers of bacteriochlorophyll and related molecules. The expressions explicitly consider the mixing of bacteriochlorophyll's four main excited states (Q_y, Q_x, B_x and B_y) in the ground and excited states of the dimer. This mixing must be considered in order to account for the hyperchromism and nonconservative circular dichroism found experimentally in oligomers of bacteriochlorophyll and bacteriopheophytin. The spectroscopic properties of the eight absorption bands of a bacteriopheophytin dimer are calculated as functions of the geometry of the dimer. The importance of the mixing of nondegenerate excited states, and of the mixing of doubly-excited states into the dimer's ground state, is evaluated by comparisons with calculations in which these phenomena are neglected. Structures for bacteriopheophytin dimers are found for which most of the calculated spectroscopic properties are consistent with the properties seen experimentally. Possible explanations are considered for the remaining discrepancies between the calculated and observed properties.     

The oxidation of chlorophyll a in alcohols

Oxidation potentials for the chlorophyll (Chl/Chl⁺) couple, relative to that of the Fe²⁺/Fe³⁺ couple, were determined in methanol, ethanol, and isopropanol from the initial extent of reaction of chlorophyll with FeCl₃. The presence of 4,7-dimethylphenanthroline was necessary to get sufficient oxidation of chlorophyll in isopropanol. The values were +0.75 V, +0.865 V, and approx. +1.0 V, respectively, based on an assumed value of +0.77 V for the Fe²⁺/Fe³⁺ potential. It is suggested that alcohols, especially methanol, may stabilize Chl⁺ by adding to the carbonyl group or the conjugated double bond system.     

Electron transfer in reaction center core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum

Electron transfer in reaction center core (RCC) complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium tepidum was studied by measuring flash-induced absorbance changes. The first preparation contained approximately three iron-sulfur centers, indicating that the three putative electron acceptors F(X), F(A), and F(B) were present; the Chl. tepidum complex contained on the average only one. In the RCC complex of Ptc. aestuarii at 277 K essentially all of the oxidized primary donor (P840(+)) created by a flash was rereduced in several seconds by N-methylphenazonium methosulfate. In RCC complexes of Chl. tepidum two decay components, one of 0.7 ms and a smaller one of about 2 s, with identical absorbance difference spectra were observed. The fast component might be due to a back reaction of P840(+) with a reduced electron acceptor, in agreement with the notion that the terminal electron acceptors, F(A) and F(B), were lost in most of the Chl. tepidum complexes. In both complexes the terminal electron acceptor (F(A) or F(B)) could be reduced by dithionite, yielding a back reaction of 170 ms with P840(+). At 10 K in the RCC complexes of both species P840(+) was rereduced in 40 ms, presumably by a back reaction with F(X)(-). In addition, a 350 micros component occurred that can be ascribed to decay of the triplet of P840, formed in part of the complexes. For P840(+) rereduction a pronounced temperature dependence was observed, indicating that electron transfer is blocked after F(X) at temperatures below 200 K.     

Coordinate expression of light-harvesting chlorophyll a/b gene family of photosystem II and chlorophyll a oxygenase gene regulated by salt-induced phosphorylation in Dunaliella salina

As a stress factor, salt induces the phosphorylation of light-harvesting chlorophyll (Chl) a/b proteins (LHCII) in Dunaliella salina. In this study, we found that the salt-induced phosphorylation of LHCII was not affected by phosphatase, and that salt simultaneously regulated both the phosphorylation of LHCII and the expression of genes encoding light-harvesting Chl a/b proteins of photosystem II (lhcb) and the gene encoding Chl a oxygenase (cao) in dark-adapted D. salina. The mRNA accumulation patterns of lhcb and cao were similar, which further affected the size of LHCII and the ratio of Chl a to Chl b. Therefore, we inferred this simultaneous regulation is one of the mechanisms of D. salina to adapt to the high-salinity environment.     

Evidence for 5- and 6-coordinated magnesium in bacteriochlorophyll a from visible absorption spectroscopy

The visible absorption spectrum of bacteriochlorophyll a (Bchl) in the 560–620 nm (yellow) region can be used to establish the coordination state of the central Mg atom. Five-coordinated Mg species absorb near 580 nm, whereas 6-coordinated Mg species are red-shifted to about 610 nm. Five-coordination is confirmed to be the principal coordination state of Mg in self-aggregated bacteriochlorophyll. The equilibrium constant for the reaction Bchl · Py + Py ai Bchl · Py₂ has been determined from computer-assisted analyses of spectral data, where Py represents pyridine. The spectral criteria for the coordination state of Mg in bacteriochlorophyll advanced here are shown to be applicable to both in vitro and in vivo systems. Similar spectral behavior is exhibited by chlorophylls a and b, and a band at 633 nm is shown to be associated with the presence of 6-coordinated Mg in chlorophyll a.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

Phylogeny of the PscB reaction center protein from green sulfur bacteria

The reaction center (RC) of green sulfur bacteria has iron—sulfur clusters as terminal acceptors and is related to the Type I RC found in Heliobacter sp. and in Photosystem I (PS I) of green plants and cyanobacteria. Degenerate primers were used to retrieve the genes coding for one of the RC proteins, PscB, from 11 strains of green sulfur bacteria. PCR using the same primers gave no product with a second group of strains and the protein from these strains did not crossreact with antibodies raised against purified PscB from the first group, suggesting the presence of a high degree of variability. The sequences shared a high degree of similarity in the region coding for the binding motif for the 4Fe–4S centers. However, the N-terminal portion of the deduced protein sequences was highly variable and contained a highly positively charged, low-complexity region with repeated tetrapeptides with two alanines flanked by proline or lysine. The PscB sequences obtained fell into two major groups, and the results suggested a lack of correlation between the pigmentation of the chlorosome antenna system and the reaction center protein. There is also a lack of correlation between the grouping of the pscB sequences and the phylogeny deduced from 16S rRNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

Calcium-induced formation of chlorophyll b and light-harvesting chlorophyll a/b-protein complex in cucumber cotyledons in the dark

Dark-grown cucumber seedlings were exposed to intermittent light (2 min light and 98 min dark) and then cotyledons were incubated with 50 mM CaCl₂ in the dark. Chlorophyll (Chl) a was selectively accumulated under intermittent light and Chl b was accumulated during the subsequent dark incubation with CaCl₂. The change in chlorophyll-protein complexes during Chl b accumulation induced by CaCl₂ in the dark was investigated by SDS-polyacrylamide gel electrophoresis. Chlorophyll-protein complex I and free chlorophyll were major chlorophyll-containing bands of the cotyledons intermittently illuminated 10 times. When these cotyledons were incubated with CaCl₂ in the dark, the light-harvesting Chl a/b-protein complex was formed. When the number of intermittent illumination periods was extended to 55, small amounts of Chl b and light-harvesting Chl a/b-protein complex were recognized at the end of intermittent light treatment, and these two pigments were further increased during the subsequent incubation of the cotyledons with CaCl₂ in the dark compared to water controls.     

Atomic coordinates for the chlorophyll core of a bacteriochlorophyll a-protein from green photosynthetic bacteria

Preliminary atomic coordinates are presented for the seven bacteriochlorophylls constituting the core of one subunit of the trimeric water soluble bacteriochlorophyll $\text{a}$-protein from the green photosynthetic bacterium $\text{Prosthecochloris aestuarii}$, strain 2K, formerly identified as $\text{Chlorobium limicola}$, strain 2K. The coordinates were derived from a 2.8 Å resolution electron density map based on four isomorphous heavy-atom derivatives, and adjusted to have stereochemically acceptable bond lengths and angles.     

Linear dichroism of electric field oriented bacteriochlorophyll a-protein from green photosynthetic bacteria

Bacteriochlorophyll a-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Q_y region of the bacteriochlorophyll a absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The ≈12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll a-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Q_y exciton transition observed in absorption at the same wavelength.     

Arrangement of the light-harvesting chlorophyll a/b protein complex in the thylakoid membrane

The transverse orientation of the light-harvesting chlorophyll a/b protein complex of Photosystem II (LHC II) in the thylakoid membrane of pea was investigated using surface radioiodination with Iodo-Gen^TM. The labelling effects on LHC II of four different membrane preparations were compared. One preparation was oriented right-side-out (intact thylakoids); two of them had an inside-out orientation exposing the lumenal surface (inside-out vesicles; PS II particles) and one had both sides of the membrane exposed (mechanically damaged thylakoids). It was found that LHC II could be iodinated only in membrane preparations with an exposed lumenal surface. Isolated apoproteins were chemically cleaved. Fragments analysis revealed a tyrosine residue located eight amino acids from the C-terminus as the single iodination site. It is concluded that the C-terminus of LHC II points towards the lumental side of the thylakoid. Differences in the labelling behaviour of the LHC apoproteins could be assigned to a heterogeneity in the C-terminal region in which the tyrosine residue is replaced by phenylalanine.     

Effect of reduction of the reaction center intermediate upon the picosecond oxidation reaction of the bacteriochlorophyll dimer in Chromatium vinosum and Rhodopseudomonas viridis

The photo-oxidation of the reaction center bacteriochlorophyll dimer or special pair was monitored at 1235 nm in Chromatium vinosum and at 1301 nm in Rhodopseudomonas viridis. In both species, the photo-oxidation was apparently complete within 10 ps after light excitation and proceeded unimpeded at low temperatures regardless of the prior state of reduction of the traditional primary electron acceptor, a quinone-iron complex. Thus the requirement for an intermediary electron carrier (I), previously established by picosecond measurements in Rps. sphaeroides (see ref. 4), is clearly a more general phenomenon.

The intermediary carrier, which involves bacteriopheophytin, was examined from the standpoint of its role as the direct electron acceptor from the photo-excited reaction center bacteriochlorophyll dimer. To accomplish this, the extent of light induced bacteriochlorophyll dimer oxidation was measured directly by the picosecond response of the infrared bands and indirectly by EPR assay of the triplet/biradical, as a function of the state of reduction of the I/I couple (measured by EPR) prior to activation. Two independent methods of obtaining I in a stably reduced form were used: chemical equilibrium reduction, and photochemical reduction. In both cases, the results demonstrated that the intermediary carrier, which we designate I, alone governs the capability for reaction center bacteriochlorophyll photooxidation, and as such I appears to be the immediate and sole electron acceptor from the light excited reaction center bacteriochlorophyll dimer.     

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Isolation, characterization, and comparison of a ubiquitous pigment-protein complex consisting of a reaction center and light-harvesting bacteriochlorophyll proteins present in purple photosynthetic bacteria

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.