Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 s for the B800-850 complex, 14±2 s for the B850 complex incorporated with spheroidene, and 19±2 s for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 s. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.     

Femtosecond energy-transfer processes in the B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1

The B800-to-B850 energy transfer time in the purified B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1 is determined to be 0.7 ps at room temperature. The electronic state dynamics of the principal carotenoid of this species, spheroidene, are examined, both in vivo and in vitro, by direct femtosecond time-resolved experiments and by fluorescence emission yield studies. Evidence is presented which suggests that carotenoid-to-bacteriochlorophyll energy transfer may occur directly from the initially excited carotenoid S2 state, as well as from the carotenoid S1 state. Further support for this conjecture is obtained from calculations of energy transfer rates from the carotenoid S2 state. Previous measurements of in vivo carotenoid and B800 dynamics are discussed in light of the new results, and currently unresolved issues are described.     

Phosphatidylcholine is required for the efficient formation of photosynthetic membrane and B800-850 light-harvesting complex in Rhodobacter sphaeroides

No phosphatidylcholine (PC) was detected in the membrane of Rhodobacter sphaeroides pmtA mutant (PmtA1) lacking phosphatidylethanolamine (PE) N-methyltransferase, whereas PE in the mutant was increased up to the mole % comparable to the combined level of PE and PC of wild type. Neither the fatty acid composition nor the fluidity of membrane was altered by pmtA mutation. Consistently, aerobic and photoheterotrophic growth of PmtA1 were not different from wild type. However, PmtA1 showed an extended lag phase (15 h) after the growth transition from aerobic to photoheterotrophic conditions, indicating the PC requirement for the efficient formation of intracytoplasmic membrane (ICM). Interestingly, the B800-850 complex of PmtA1 was decreased more than twofold in comparison with wild type, whereas the level of the B875 complex comprising the fixed photosynthetic unit was not changed. Since puc expression is not affected by pmtA mutation, PC appears to be required for the proper formation of the B800-850 complex in the ICM of R. sphaeroides.     

The effect of different levels of the B800-850 light-harvesting complex on intracytoplasmic membrane development in Rhodobacter sphaeroides

A role for the peripheral (B800-850) light-harvesting complex in vesicularization of the Rhodobacter sphaeroides intracytoplasmic membrane (ICM), suggested from studies in mutant strains lacking one or more of the pigment-protein complexes, was examined further in the wild-type strain NCIB 8253 grown at high (∼1000 W m^–2), moderate (∼300 W m^–2), and low (∼100 W m^–2) light intensities. The resulting ICM vesicles (chromatophores) had B800-850 levels related inversely to irradiance and banded in rate-zone sedimentation at ∼1.10, 1.09, and 1.07 g ml^–1, respectively. Equilibrium centrifugation on iso-osmotic gradients indicated that this distinct sedimentation behavior resulted solely from differences in hydrodynamic radii. These size differences were confirmed by gel-exclusion chromatography and in electron micrographs of thin-sectioned cells. A pulse-chase study of ICM growth following a tenfold reduction in light intensity showed a relatively slow equilibration of membrane proteins during adaptation, and that new protein was incorporated largely into additional ICM formed at the lowered illumination level, giving rise to chromatophores of reduced size and elevated B800-850 content. These results provide further evidence for a model in which the B800-850 complex both drives development of vesicular ICM in Rba. sphaeroides and determines the size of resulting vesicles. Received: 12 October 1995 / Accepted: 21 December 1995     

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800–850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a free pigment band; the remaining absorption appeared at 880 nm and shifted to 845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min^-1 for the initial rapid change and 0.05 and 0.11 min^-1 for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.Abbreviations BChl bacteriochlorophyll a - BPheo bacteriopheophytin a     

Molecular organization of bacteriochlorophyll in the light-converging B850 complex of purple bacteria

The light-harvesting bacteriochlorophyll-protein complex B850 has been isolated from two species of purple bacteria, Rhodopseudomonas palustris and Chromatium minutissimum. Absorption and fluorescence spectra at 20 degrees and--196 degrees C of this complex were registered. Second derivatives of the absorption spectra, Stepanov's relation and computer curve analyses in terms of asymmetrical Gaussian components show that absorption spectra consist of five and fluorescence spectra--of three components. These components were analysed in terms of exciton interaction among bacteriochlorophyll molecules. Data obtained were used for building-up of the molecular model of the complex.     

Energy transfer between the carotenoid and the bacteriochlorophyll within the B-800-850 light-harvesting pigment-protein complex of Rhodopseudomonas sphaeroides

Energy transfer between carotenoid and bacteriochlorophyll has been studied in isolated B-800-850 antenna pigment-protein complexes from different strains of Rhodopseudomonas sphaeroides which contain different types of carotenoid. Singlet-singlet energy transfer from the carotenoid to the bacteriochlorophyll is efficient (75-100%) and is rather insensitive to carotenoid type, over the range of carotenoids tested. The yield of carotenoid triplets is low (2-15%) but this arises from a low yield of bacteriochlorophyll triplet formation rather than from an inefficient triplet-triplet exchange reaction. The rate of the triplet-triplet exchange reaction between the bacteriochlorophyll and the carotenoid is fast (Ktt greater than or equal to 1.4 . 10(8) S-1) and also relatively independent of the type of carotenoid present.     

The light-harvesting polypeptides of Rhodopseudomonas sphaeroides R-26.1. II. Conformational analyses by attenuated total reflection infrared spectroscopy and the possible molecular structure of the hydrophobic domain of the B 850 complex

Attenuated total reflection infrared spectroscopy were used to study the conformation of the purified light-harvesting polypeptides from Rhodopseudomonas sphaeroides R-26.1. B 870-alpha and B 850-beta are characterised by a high content of alpha-helix; B 850-alpha and B 870-beta, in contrast, contain extensive antiparallel chain-pleated sheet structure. The beta-structure is likely to be an artifact of the isolation because B 850-alpha assumes a predominantly alpha-helical conformation in the intact antenna complex. It is concluded that lipid-protein interactions play a crucial role in the stabilisation of the "native" alpha-helical fold of B 850-alpha and thus in the stabilisation of the entire antenna-pigment-protein complex. The results obtained concerning the "in situ" conformation of B 850-alpha and B 850-beta were used, together with the known primary structures and data available from the literature, to construct a rough molecular model of the hydrophobic domain of the elementary unit of the B 850 complex.     

Model for the light-harvesting complex I (B875) of Rhodobacter sphaeroides.

The light-harvesting complex I (LH-I) of Rhodobacter sphaeroides has been modeled computationally as a hexadecamer of alphabeta-heterodimers, based on a close homology of the heterodimer to that of light-harvesting complex II (LH-II) of Rhodospirillum molischianum. The resulting LH-I structure yields an electron density projection map that is in agreement with an 8.5-A resolution electron microscopic projection map for the highly homologous LH-I of Rs. rubrum. A complex of the modeled LH-I with the photosynthetic reaction center of the same species has been obtained by a constrained conformational search. This complex and the available structures of LH-II from Rs. molischianum and Rhodopseudomonas acidophila furnish a complete model of the pigment organization in the photosynthetic membrane of purple bacteria.     

Influence of carotenoid molecules on the structure of the bacteriochlorophyll binding site in peripheral light-harvesting proteins from Rhodobacter sphaeroides

In the LH2 proteins from Rhodobacter (Rb.) sphaeroides, the hydrogen bonds between the bacteriochlorophyll (Bchl) molecules and their proteic binding sites exhibit a strong variance with respect to carotenoid content and type. In the absence of the carotenoid molecule, such as in the LH2 from Rb. sphaeroides R26.1, the void in the protein structure induces a significant reorganization of the binding site of both Bchl molecules responsible for the 850 nm absorption, which is not observed when the 800 nm absorbing Bchl is selectively removed from these complexes. FT Raman spectra of LH2 complexes from Rb. sphaeroides show that the strength of the hydrogen bond between the 850 nm absorbing Bchl bound to the alpha polypeptide and the tyrosine alpha(45) depends precisely on the chemical nature of the bound carotenoid. These results suggest that the variable extremity of the carotenoid is embedded in these LH2 complexes, lying close to the interacting Bchl molecules. In the LH2 from Rhodopseudomonas acidophila, the equivalent part of the rhodopin glucoside, which bears the glucose group, lies close to the amino terminal of the antenna polypeptide. This contrast suggests that the structure of the carotenoid binding site in LH2 complexes strongly depends on the bacterial species and/or on the chemical nature of the bound carotenoid.     

Intracytoplasmic membrane vesiculation in light-harvesting mutants of Rhodobacter sphaeroides and Rhodobacter capsulatus

Abstract In Chlamydomonas reinhardtii there are three glutamate dehydrogenase isozymes which can use both NADH and NADPH as cofactors and respond differently to different nitrogen sources and several stress conditions. From data of induction of isozymes in different metabolic situations, we propose a possible physiological role for each of them in algal carbon and nitrogen metabolism.     

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl?d, Chl?f or bacteriochlorophyll (BChl) b to replace native BChl?a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg_?10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6?ps, energy transfer from Chl?a to B850 BChl?a remained highly efficient. We measured faster energy-transfer time constants for Chl?d (3.5?ps) and Chl?f (2.7?ps), which have red-shifted absorption maxima compared to Chl?a. BChl?b, red-shifted from the native BChl?a, gave extremely rapid (≤0.1?ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range.     

Transverse membrane topography of the B875 light-harvesting polypeptides of wild-type Rhodobacter sphaeroides.

Purified B875 light-harvesting complex, chromatophores, and spheroplast-derived vesicles from wild-type Rhodobacter sphaeroides were treated with proteinase K or trypsin, and the alpha and beta polypeptides were analyzed by electrophoretic, immunochemical, and protein-sequencing methods. With the purified complex, proteinase K digested both polypeptides and completely eliminated the A875 peak. Trypsin digested the alpha polypeptide and reduced the A875 by 50%. Proteinase K cleaved the beta polypeptide of chromatophores and the alpha polypeptide of spheroplast-derived vesicles. Sequence analyses of polypeptides extracted from proteinase K-treated chromatophores revealed that the beta polypeptide was cleaved between amino acids 4 and 5 from the N terminus. The N terminus of the alpha polypeptide was intact. We concluded that the N terminus of the beta polypeptide is exposed on the cytoplasmic membrane surface, and the difference in the digestion patterns between the spheroplast-derived vesicles and chromatophores suggested that the C terminus of the alpha polypeptide is exposed on the periplasmic surface.     

Carotenoid triplet state formation in Rhodobacter sphaeroides R-26 reaction centers exchanged with modified bacteriochlorophyll pigments and reconstituted with spheroidene

Triplet state electron paramagnetic resonance (EPR) experiments have been carried out at X-band on Rb. sphaeroides R-26 reaction centers that have been reconstituted with the carotenoid, spheroidene, and exchanged with 13²-OH-Zn-bacteriochlorophyll a and [3-vinyl]-13²-OH-bacteriochlorophyll a at the monomeric, accessory bacteriochlorophyll sites B_A,B or with pheophytin a at the bacteriopheophytin sites H_A,B. The primary donor and carotenoid triplet state EPR signals in the temperature range 95–150 K are compared and contrasted with those from native Rb. sphaeroides wild type and Rb. sphaeroides R-26 reaction centers reconstituted with spheroidene. The temperature dependencies of the EPR signals are strikingly different for the various samples. The data prove that triplet energy transfer from the primary donor to the carotenoid is mediated by the monomeric, BChl_B molecule. Furthermore, the data show that triplet energy transfer from the primary donor to the carotenoid is an activated process, the efficiency of which correlates with the estimated triplet state energies of the modified pigments.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - Chl chlorophyll - EPR electron paramagnetic resonance - LDAO lauryl-dimethylamine-N-oxide - Phe pheophytin