Generation of triplet and cation-radical bacteriochlorophyll a in carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides

The LH1 antenna complex and a native form of the LH2 complex were isolated from the carotenoidless R26 and R26.1 mutants of Rhodobacter sphaeroides by the use of a new detergent, sucrose monocholate. One-color, pump-and-probe transient Raman spectroscopy of these complexes using 351 nm, approximately 50 ps pulses showed the generation of the triplet state of bacteriochlorophyll a (BChl a), whereas measurements using 355 nm, approximately 12 ns pulses showed the generation of BChl a cation radical. Subpicosecond to nanosecond time-resolved absorption spectroscopy using 388 nm, 200 fs pulses for excitation showed rapid (<1 ps) generation of the triplet state and fast decay (<10 ps) of the singlet state of BChl a. Microsecond absorption spectroscopy confirmed the generation of BChl a cation radical. EPR spectroscopy using 532 nm, approximately 5 ns pulses for excitation established the generation of BChl a cation radical. The EPR line width suggested that the unpaired electron is shared by two BChl a molecules. In LH1, the yield of BChl a cation radical per complex was estimated to be about 80% of that in the reaction center, and in LH2 about 50%. Thus, rapid generation of the triplet state, and its subsequent transformation into the cation-radical state of BChl a have been shown to be intrinsic properties of B870 and B850 BChl a assembly in the carotenoidless LH1 and LH2 antenna complexes. In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex is shown.     

Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides

We have examined mutants in the core light-harvesting complex of Rhodobacter sphaeroides in which the tryptophan residues located at positions alpha+11, beta+6, and beta+9 have been mutated to each of the three other aromatic amino acids, namely tyrosine, phenylalanine, and histidine. We confirm that the alpha+11 residue and show that the beta+9 residue each form a hydrogen bond to a C2-acetyl group of a BChl molecule. Mutation of either of these residues to a phenylalanine results in a breakage of the normal hydrogen bond, whereas a histidine in either of these positions is able to form a hydrogen bond to the BChl. Comparison of the absorption spectra with the hydrogen bonding of the C2-acetyl groups for the various mutants demonstrates a role for this molecular interaction in the tuning of the absorption properties of the complex. We further demonstrate that there is a consistent linear relationship between the downshift in the C2-acetyl stretching mode and the red shift in the absorption maximum, in both core and peripheral antenna complexes. This linear relationship allows us to estimate the contribution of H bonding to the red shifts of these complexes. Though the residue beta+6 is found not to be directly involved in interactions with the pigment molecules, mutation of this residue is shown in some cases to result in both a destabilization of the complex and a decrease in the binding site homogeneity. Finally, a consideration of the amount of antenna complex present in the various mutants shows an important role for the reaction center and/or the pufX gene product in the assembly or stabilization of this membrane protein.     

Ultrafast exciton relaxation in the B850 antenna complex of Rhodobacter sphaeroides

Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacterium Rhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.     

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.     

Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.     

Identification of a methyl-accepting chemotaxis protein in Rhodobacter sphaeroides

Pyocin typing may be unstable under certain circumstances. In the early seventies, it was shown that the IncP-2 plasmids pMG1 and pMG2 interfered with the pyocin type of some Pseudomonas aeruginosa strains. Up to now, details of the influence of these plasmids on the pyocins are unknown. In this study, the effect of these plasmids on the R, F, and S pyocins, respectively, was examined. In distinct strains, S pyocin activity was detected in the presence of these plasmids, whereas R and F pyocin activities could not be observed. So the effect of the IncP-2 plasmids pMG1 and pMG2 may be limited to the R and/or F pyocins.     

Photosynthetic deficiency of a pufX deletion mutant of Rhodobacter sphaeroides is suppressed by point mutations in the light-harvesting complex genes pufB or pufA

The pufX gene of the facultative phototroph Rhodobacter sphaeroides encodes a membrane protein that is required for photoheterotrophic growth. Deletion of pufX impairs the photosynthetic generation of a transmembrane potential, suggesting a role for the PufX protein in light-driven cyclic electron transfer [Farchaus, J. W., et al. (1992) EMBO J. 11, 2779-2788]. Here we describe the isolation and characterization of 65 spontaneous suppressor mutants in which photosynthetic competence was restored by secondary mutations. Genetic analysis revealed the occurrence of single point mutations altering highly conserved residues within the light-harvesting complex, B875. One of three tryptophan codons was changed to stop or arginine codons in 89% of these suppressor mutants. Spectral characterization and Western blot analysis were used to examine the B875 assembly and the stable expression of the altered light-harvesting polypeptides. Three different groups of suppressor mutants were found: (1) No stable expression of altered B875 polypeptides was detected for the alpha 43W-->* and beta 44W-->* mutants. (2) There was expression of the mutated B875-beta chain, but no stable B875 assembly in the beta 47W-->R mutants. (3) Intact B875 complexes were found for the alpha 47S-->F or beta 20H-->R mutants. These results provide evidence that the differently altered B875 polypeptides do not substitute directly for the PufX protein but lead to structural rearrangements in the macromolecular membrane organization, thus restoring a sufficiently high capacity for light-driven cyclic electron transfer.     

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.     

Identification of the upper exciton component of the B850 bacteriochlorophylls of the LH2 antenna complex, using a B800-free mutant of Rhodobacter sphaeroides

In this paper, we report the circular dichroism (CD) spectra of two types of LH2-only mutants of Rhodobacter sphaeroides. In the first, only the wild type LH2 is present, while i the second, the B800 binding site of LH2 has been either destabilized or removed. For the first time, we have identified a band in the CD spectrum of LH2, located at approximately 780 nm, that can be ascribed to the high exciton component of the B850 band. The experimental spectra have been modeled by theoretical calculations. On this basis, the average interaction strength between the monomers in the B850 ring can be estimated to be approximately 300 cm-1. In addition, we suggest that in LH2 of Rb. sphaeroides the angles made by the Qy transitions of the B850 BChls with respect to the plane of the ring are slightly different from those calculated from the crystal structure of the Rhodopseudomonas acidophila LH2 complex.     

A conformational change of the photoactive bacteriopheophytin in reaction centers from Rhodobacter sphaeroides

It is demonstrated by ENDOR and Special TRIPLE spectroscopy that two distinct radical anion states of the intermediate electron acceptor (I), a bacteriopheophytin, can be freeze-trapped in isolated photosynthetic reaction centers of Rhodobacter sphaeroides. The formation of these states depends on the illumination time prior to freezing and the temperature. The first state, I1.-, is metastable and relaxes irreversibly at T approximately 160 K to the second state, I2.-. Experiments on quinone depleted as well as mutant reaction centers help to exclude the possibility that other cofactors besides the bacteriopheophytin in the A-branch, PhiA, are reduced during the trapping procedure. In particular, two mutants are investigated, in which the hydrogen bonds to PhiA that exist in the wild type are removed. These mutants are EL(L104), in which Glu at position L104 near the 13(1)-keto group of PhiA is replaced by Leu, and WF(L100), in which Trp at position L100 near the 13(2)-methyl ester of PhiA is replaced by Phe. Both mutations have characteristic effects on both I.- states. In addition, the replacement of Thr at position M133 near the 13(1)-keto group of the inactive bacteriopheophytin and of Gly at position M203 near the 13(1)-keto group of the accessory bacteriochlorophyll in the A-branch by Asp causes no changes of the electronic structure of I.-. The two I.- states are interpreted in terms of a reorientation of the 3-acetyl group of PhiA after reduction. Possible implications for the initial charge separation process are discussed.     

Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c2 complex from Rhodobacter sphaeroides

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.     

The primary structures of the low-redox potential diheme cytochromes c from the phototrophic bacteria Rhodobacter sphaeroides and Rhodobacter adriaticus reveal a new structural family of c-type cytochromes

The complete amino acid sequence of the low-redox potential cytochrome c-551.5 from Rhodobacter sphaeroides was determined by automated Edman degradation combined with mass spectroscopy. There are 139 residues and two typical Cys-X-X-Cys-His heme-binding sites. A homologous low-redox potential cytochrome was also sequenced from Rhodobacter adriaticus and was found to contain 126 residues. It is 53% identical to that of Rb. sphaeroides and has two internal deletions of one and five residues. The Rhodobacter diheme cytochromes are 21-24% identical to the translated open reading frame SLL1886 from Synechocystis sp. PCC6801. There are at least two deletions of five and eight residues in the 188-residue cyanobacterial protein. Each of the three cytochromes has more histidines than it needs to bind the two hemes, but conserved histidines located 23 residues after the first heme and 14-19 residues before the second heme are likely to be the sixth heme ligands. There is no evidence for gene doubling and no similarity to any other known cytochromes. The measured helix content of 24% is much less than normal for c-type cytochromes. These proteins thus appear to be representative of an entirely new class of c-type cytochromes.     

Plasmid content and localization of the genes encoding the denitrification enzymes in two strains of Rhodobacter sphaeroides

In rats injected with neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) the development of experimental depressive syndrome was accompanied by local epileptiform activity in the caudate-putamen complex and by reorganization of electrical processes in the brain. The spectral power density in the caudate-putamen in the delta range was increased in the formative stage of depressive syndrome (day 3-4 from the beginning of MPTP administration) and in the stage of behaviour recovery (a week after the withdrawal) as compared to control rats. On the contrary, the spectral power in the alpha range was decreased at the peak of depression (day 11-12 from the beginning of neurotoxin administration) and a week after the withdrawal as compared to the initial value. In the formative stage of depressive syndrome the spectral power in the delta range was increased in hippocampus whereas in sensorimotor cortex it was decreased at the frequency 6 Hz compared to control. It is suggested that a new pathodynamical organization is formed in the CNS of animals in response to MPTP administration, which is thought to be a neuropathophysiological basis of depressive syndrome.     

New crystal form of the photosynthetic reaction centre from Rhodobacter sphaeroides of improved diffraction quality

Trigonal crystals of photosynthetic reaction centres from the wild-type purple bacterium, Rhodobacter sphaeroides (ATCC 17023), have been grown from potassium phosphate solutions at 18 degrees C. They belong to the space group P3(1/2)21 and have unit cell dimensions of a = b = 141.4 A and c = 187.2 A. The crystals diffract to at least 2.65 A resolution and are suitable for detailed structural studies.     

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.     

Genetic evidence for the role of isocytochrome c2 in photosynthetic growth of Rhodobacter sphaeroides Spd mutants

In Rhodobacter sphaeroides, cytochrome c2 (cyt c2)-deficient mutants are photosynthetically incompetent (PS-). However, mutations which suppress the photosynthetic deficiency (spd mutations) of cyt c2 mutants increase the levels of a cyt c2 isoform, isocyt c2. To determine whether isocyt c2 was required for photosynthetic growth of Spd mutants, we used Tn5 mutagenesis to generate a PS- mutant (TP39) that lacks both cyt c2 and isocyt c2. DNA sequence analysis of wild-type DNA that restores isocyt c2 production and photosynthetic growth to TP39 indicates that it encodes the isocyt c2 structural gene, cycI. The Tn5 insertion in TP39 is approximately 1.5 kb upstream of cycI, and our results show that it is polar onto cycI. The cycI gene has been physically mapped to a region of chromosome I that is approximately 700 kb from the R. sphaeroides photosynthetic gene cluster. Construction of a defined cycI null mutant and complementation of several mutants with the cycI gene under the control of the cyt c2 promoter region indicate that an increase in the levels of isocyt c2 alone is necessary and sufficient for photosynthetic growth in the absence of cyt c2. The data are discussed in terms of the obligate role of isocyt c2 in cyt c2-independent photosynthesis of R. sphaeroides.     

Expression of glnB and a glnB-like gene (glnK) in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant of Rhodobacter sphaeroides

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.