BChl Molecules Research Articles

Formation of the light-harvesting complex I (B870) of anoxygenic phototrophic purple bacteria.

The light-harvesting (LH) complex I (B870) of anoxygenic photosynthetic purple bacteria is the oligomeric form of its subunit B820 consisting of the low-molecular-weight polypeptides alpha, beta, bacteriochlorophyll (BChl), and carotenoids in the stoichiometric ratio [alpha1 beta1 (BChl2) Crt1-2]n. LHI surrounds the photochemical reaction center (RC). The major absorption band of the LHI complex is species-specific and is found at 870-890 nm; those of the subunit and the monomeric BChl a (dissolved in methanol) absorb at 820 and 770 nm, respectively. The isolated LHI complex can be reversibly dissociated to the B820 subunit or to the polypeptides and pigments by addition of detergents. Reconstitution of the B820 or the functional B870 complex is still possible after partial truncation of the N- or C-terminal regions of the alpha- or beta-polypeptide or of the beta-polypeptide only. The minimal structural requirements for reconstitution of a spectrally wild-type form after truncation of the polypeptides and/or modifications of the BChl molecule are described. The insertion of the LHIalpha- and LHIbeta-polypeptides into the membrane and the in vivo assembly of LHI, studied in a cell-free system and in whole cells of Rhodobacter capsulatus, depend on the primary structures of both polypeptides, BChl, the chaperones DnaK and GroEL, membrane-bound proteins, and energized membranes. Exchanges, deletions, or insertions of amino acyl residues, especially in the conserved region of the N-terminus of the LHIalpha-polypeptide, prevent or reduce the efficiency and stability of the LHI assembly. Therefore, reconstitution of LHI in a detergent micelle does not exactly reproduce the formation of the LHI complex in the photosynthetic membrane in vivo. The N-terminal domains play a crucial role in the formation of the oligomeric protein scaffold and of the pigment array. Facultatively phototrophic bacteria such as Rhodospirillum (Rsp.) rubrum or Rhodobacter (Rba.) capsulatus can adjust to changes in oxygen tension, light intensity, temperature, and substrates to grow under chemotrophic or phototrophic conditions. The photosynthetic apparatus (PSA), localized mainly on intracytoplasmic membranes (ICM), is usually synthesized only under low oxygen partial pressure. The cellular amount and composition of the PSA are modified upon changing light intensity in relation to cell growth (Drews and Golecki 1995). The morphogenesis of cellular structures like ICM is quite different from self-assembly. Self-assembly is a reversible process of aggregation of the constituents of a complex structure without protein synthesis and is driven by weak or strong forces in the interactions of the constituents. Morphogenesis results from the interplay of numerous gene products and the cellular organization and is always dependent upon pre-existent structures (Harold 1995). The morphogenesis of the photosynthetic membrane in purple bacteria has been studied in its different steps. The regulation at the transcriptional and post-transcriptional levels in purple bacteria, and the structure and morphogenesis of the ICM have been described recently (Armstrong 1995; Bauer 1995; Biel 1995; Drews and Golecki 1995; Klug 1995). In this mini-review, I will focus on the minimal requirements for the in vitro assembly of light-harvesting (LH) complex I (B870) from its constituents in detergent micelles and compare the results with observations on the complex process of targeting and import of LHI polypeptides into the membrane and assembly of B870.

Structure‐Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll‐Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light‐harvesting complex 2 (LH2) bacteriochlorophyll‐protein antenna complex from the purple non‐sulfur photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure‐based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near‐infrared region use a point‐monopole approximation to represent the BChl Qy transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane‐associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm−1, respectively) for the Qy transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm−1 wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.

Excitations and excitons in bacterial light-harvesting complexes

The arrangement of pigments as recently observed in the structure of the peripheral light-harvesting complex of Rhodopseudomonas acidophila provides a new impulse to the discussion about the nature of the excitation transport and exciton delocalization in these photosynthetic light-harvesting systems. A crucial parameter in this discussion is the strength of the coupling between neighbouring BChl molecules. Their proximity implies that exciton interaction is important in such a structure, and this tends to delocalize the excitation. However, at the same time these structures exhibit strong spectral inhomogeneity, due to which the excitation becomes localized. In this paper we discuss some views on the organization, interactions and energy transfer dynamics in light-harvesting complexes of photosynthetic purple bacteria.

Electronic structure of the oxidized primary electron donor of the HL (M202) and HL (L173) heterodimer mutants of the photosynthetic bacterium Rhodobacter sphaeroides: ENDOR on single crystals of reaction centers

The electronic structure of the primary electron donor (D) in the heterodimer mutants mutants HL (M202) and HL (L173) of the photosynthetic bacterium Rhodobacter sphaeroides was investigated using EPR and ENDOR (electron nuclear double resonance) methods on single crystals of reaction centers. In the mutants, one of the two bacteriochlorophyll (BChl) molecules of D is replaced by a bacteriopheophytin. The assignment of the ENDOR lines to specific methyl and non-methyl protons was accomplished by comparing that directions of the principal axes of the hyperfine tensors with the directions predicted from the X-ray structure and theory. We showed that the unpaired electron is localized on the BChl in the heterodimers, i.e., on the L-side (D L) in the HL (M202) and on the M-side (D M) in the HL (L173) mutant. Significant differences in the electronic structure of D L and D M were observed; they are attributed to the protein and/or pigment environment. Possible consequences of these differences for electron transfer, e.g., unidirectionality are discussed. The inequivalence of D L and D M also shows up in the asymmetry of the electronic structure of D in the native homodimer, whose electronic structure was reinterpreted using the heterodimers as monomer models.

Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center.

Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus. FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively. The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state. Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P. The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules. The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl. The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds. From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus. The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1. Compared to Rb. sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca. 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca. 80% for Rb. sphaeroides. The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed.

Hydrogen-Bond Interactions of the Primary Donor of the Photosynthetic Purple Sulfur Bacterium Chromatium tepidum

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.

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.

Singlet−Singlet Annihilation and Local Heating in FMO Complexes

Energy transfer in a trimeric Bchl a containing FMO pigment−protein complex from the green sulfur bacterium Chlorobium tepidum has been studied by means of picosecond transient absorption spectroscopy under high-excitation conditions. At room temperature the excited state absorption spectrum of the FMO complex was found to be similar to that of noninteracting Bchl molecules in solution, which suggests that the influence of exciton coupling on the spectroscopic properties of the FMO complex at room temperature is not substantial. Analysis of the excited state relaxation kinetics in singlet−singlet annihilation conditions shows that the energy transfer from the excited monomer to another excited monomer is independent of the oxidation−reduction state of the complex and is slower than the intermonomer excitation migration rate. The difference spectrum at 77 K resembles the absorption spectrum, showing three exciton subbands. In addition to the singlet−singlet annihilation, the 7 ps rate of which is similar to that at room temperature, and to the intrinsic exciton decay, which is also temperature independent, energy redistribution between exciton states with a mean time of 26 ps is evident. This redistribution is explained as being due to local heating/cooling kinetics stimulated by the excitation pulses.

Energy transfer in spectrally inhomogeneous light-harvesting pigment-protein complexes of purple bacteria

Energy transfer within the peripheral light-harvesting antenna of the purple bacteria Rhodobacter sphaeroides and Rhodopseudomonas palustris was studied by one- and two-color pump-probe absorption spectroscopy with approximately 100-fs tunable pulses at room temperature and at 77 K. The energy transfer from B800 to B850 occurs with a time constant of 0.7 +/- 0.05 ps at room temperature and 1.8 +/- 0.2 ps at 77 K and is similar in both species. Anisotropy measurements suggest a limited but fast B800 <--> B800 transfer time (tau approximately 0.3 ps). This is analyzed as incoherent hopping of the excitation in a system of spectrally inhomogeneous antenna pigment-protein complexes, by a master equation approach. The simulations show that the measured B800 dynamics is well described as energy transfer with a characteristic average nearest-neighbor pairwise transfer time of 0.35 ps among approximately 10 Bchl molecules in a circular arrangement, in good agreement with the recent high-resolution structure of LH2. The possible presence of fast intramolecular relaxation processes within the Bchl a molecule was investigated by measurement of time-resolved difference absorption spectra and kinetics of Bchl a in solution and in low-temperature glasses. From these measurements it is concluded that fast transients observed at room temperature are due mainly to solvation processes, whereas at 77 K predominantly slower (> 10-ps) relaxation occurs.

High-field EPR on the primary electron donor cation radical in single crystals of heterodimer mutant reaction centers of photosynthetic bacteria — first characterization of the G-tensor

First high-field/high-frequency (95 GHz) EPR experiments on the cation radical of the primary electron donor (D) in single crystals of the HL (M202) heterodimer mutant of the photosynthetic bacterium Rhodobacter sphaeroides are reported. The measurements have been performed in order to determine the electronic G-tensor of D+ in the mutant and to compare it to the G-tensor of D+ in wild type (R-26). In the heterodimer mutant of the two bacteriochlorophyll (BCHl) molecules constituting D in R-26 is replaced by a bacteriopheophytin. In D2+ of the mutant the unpaired electron is localized on the BChl, thus, its G-tensor should reflect monomer properties, whereas in D+ of R-26 the unpaired electron is delocalized over the two BChl molecules. The preliminary results reported here show that directions of the principal axes of the G-tensor of D+ in HL (M202) are similar to those of R-26 investigated previously. Possible origins of this result are discussed.

Structure and properties of the bacteriochlorophyll binding site in peripheral light-harvesting complexes of purple bacteria.

In this paper, we have examined, using FT resonance Raman spectroscopy, the bacteriochlorophyll (BChl) binding sites in the peripheral light-harvesting complexes extracted from a number of purple bacterial strains. A comparison of interactions of the BChl molecules with their binding sites in these LH2 complexes, together with the primary sequences of the alpha and beta polypeptides, allows three amino acids to be proposed to be involved in the hydrogen bonding of the 9-keto carbonyl of one of the 850-nm-absorbing pair of BChl molecules. Specifically, we show that one keto carbonyl group, which is strongly hydrogen bonded in Rhodobacter sphaeroides LH2, is involved in much weaker interactions in the LH2 complexes from all the other species studied (i.e., Rhodobacter capsulatus, Rubrivivax gelatinosus, Rhodopseudomonas palustris, Rhodopseudomonas acidophila, and Rhodopseudomonas cryptolactis). This is correlated with the presence of three polar amino acids in the primary sequence of the alpha polypeptide of Rb. sphaeroides which are absent in the sequences from all the other bacteria and probably close to a chromophore. These three residues are a serine at position -4, a threonine at position +6 and another serine at position +17 (numbering relative to the conserved histidine, considered as position 0), in the alpha polypeptide of Rb. sphaeroides. Furthermore, the study of the interactions in natural B800-820 complexes shows that the two 2-acetyl groups of the 820-nm-absorbing BChl molecules are free from hydrogen-bonding interactions. In the light of previous site-selected mutagenesis studies, the lack of such hydrogen bonds seems to be a general phenomenon, associated with the 820-nm absorption of LH2 complexes, and suggests that hydrogen-bonding interactions have a precise molecular role in finely tuning the functional properties of these complexes.

Exciton states of the antenna and energy trapping by the reaction center

Forward and back energy transfer between antenna and RC in the photosynthetic apparatus of purple bacteria was studied taking into account the exciton states of the antenna. The exciton states were calculated for core antenna configuration in the form of a circular aggregate of N identical BChl molecules with the CN-symmetry. The influence of pigment inhomogeneity on the proposed exciton description of the antenna and its interaction with RC was investigated. The ratio between the rate constants of forward and back energy transfer between the exciton levels of the antenna and RC was obtained as a function of the temperature, the number of antenna BChls and the antenna exciton level position with respect to BChl special pair level of RC. A versatile analytical expression for this ratio which is independent of the BChl special pair level position and its dipole orientation was derived. The proposed model results in an irreversible excitation trapping by RC even at room temperature.

Generation and quenching of singlet molecular oxygen by aggregated bacteriochlorophyll d in model systems and chlorosomes

Both photogeneration and quenching of singlet oxygen by monomeric and aggregated (dimeric and oligomeric) molecules of bacteriochlorophyll (BChl) d have been studied in solution and in chlorosomes isolated from the green photosynthetic bacterium Chlorobium vibrioforme f. thiosulfatophilum. The yield of singlet-oxygen photogeneration by pigment dimers was about 6 times less than for monomers. Singlet oxygen formation was not observed in oligomer-containing solutions or in chlorosomes. To estimate the efficiency of singlet oxygen quenching an effective rate constant for (1)O2 quenching by BChl molecules (kq (M)) was determined using the Stern-Volmer equation and the total concentration of BChl d in the samples. In solutions containing only monomeric BChl, the kq (M) values coincide with the real values for (1)O2 quenching rate constants by BChl molecules. Aggregation weakly influenced the kq (M) values in pigment solutions. In chlorosomes (which contain both BChl and carotenoids) the kq (M) value was less than in solutions of BChl alone and much less than in acetone extracts from chlorosomes. Thus (1)O2 quenching by BChl and carotenoids is much less efficient in chlorosomes than in solution and is likely caused primarily by BChl molecules which are close to the surface of the large chlorosome particles. The data allow a general conclusion that monomeric and dimeric chlorophyll molecules are the most likely sources of (1)O2 formation in photosynthetic systems and excitation energy trapping by the long wavelength aggregates as well as (1)O2 physical quenching by monomeric and aggregated chlorophyll can be considered as parts of the protective system against singlet oxygen formation.

Thermodynamics of Membrane Polypeptide Oligomerization in Light-harvesting Complexes and Associated Structural Changes

Investigation of the equilibrium between the dissociated B777 form of the light-harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form demonstrates that the B777 form consists of bacteriochlorophyll a (BChl) bound to the α or β polypeptide chains; this binding appears to be reasonably stable at room temperature with little dissociation to free Bchl and polypeptides. Analysis of the reaction order for the B777 association reaction to form B820 shows that this reaction requires four components, presumably two α-B777 units and two β-B777 units, implying that the B820 subunit contains four Bchl molecules. Estimations of the enthalpy and entropy changes associated with the tetramerization give values of, respectively, ?175 kJ mol-1 and ?0·46 Kj mol-1 K-1. Soret resonance Raman and Fourier transform preresonance Raman spectra of Bchl in detergent together with those of the B777, B820 and native B873 forms of the light harvesting complex illustrate significant changes occurring to the environments of the C-2 acetyl groups (Fischer numbering system) during dissociation to from B820 and a loss of order in the C-9 keto environments on formation of B777. Attenuated total reflectance Fourier transform infrared absorption spectra of the three antennae forms demonstrate little perturbation of the approximately 50% α-helical secondary structure during dissociation. These observations are discussed in terms of the energetics of membrane protein folding and the structure of the light harvesting complex.

SOLVENT EFFECTS ON TRIPLET‐STATE BACTERIOCHLOROPHYLL a AS DETECTED BY TRANSIENT RAMAN SPECTROSCOPY AND THE ENVIRONMENT OF BACTERIOCHLOROPHYLL a IN THE LIGHT‐HARVESTING COMPLEX OF Rhodobacter sphaeroides R26

AbstractThe frequency of BChl that was bound to the light‐harvesting complex (LHC) of Rhodobacter sphaeroides R26 was found to be 1598 cm‐1, a result which suggests that a pair of BChl molecules form a dimer in the LHC in the T1 state.

Application of near-IR Fourier transform resonance Raman spectroscopy to the study of photosynthetic proteins

We demonstrate the application of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy using 1064 nm excitation to obtain high quality preresonance Raman spectra of bacteriochlorophyll chromophores in photosynthetic proteins from purple bacteria at room temperature without sample degradation. We present NIR FT preresonance Raman spectra of chromatophores from Rhodospirillum (Rsp.) rubrum, carotenoidless strain G9 containing bacteriochlorophyll a (BChl a) chromophores mostly from its B880 antenna complex, and the B850-800 antenna complex from Rhodobacter (Rb.) sphaeroides, 2.4.1 strain; these spectra are compared with their resonance Raman (RR) spectra obtained using 363.8 nm excitation at 30 K. For antenna complexes not containing carotenoid the FT Raman spectra are dominated by the vibrational modes of the bacteriochlorophyll chromophores with no interference from the modes of the protein or membrane. In the NIR FT Raman spectrum of the B850-800 complex from Rb. sphaeroides 2.4.1, strong contributions from the carotenoid molecule are observed to cause some interference with the 1609 cm −1 band of the BChl a molecules. We also present FT Raman spectra of reaction centers (RCs) from Rb. sphaeroides R 26 in the reduced and oxidized states of their primary electron donor (P). In the reduced state, it is estimated that ca 70% of the FT Raman spectrum arises from reduced P whose electronic absorption band is at 865 nm. With 1064 nm excitation of the RCs poised in the oxidized cation radical state of P, we have observed for the first time a Raman spectrum of P +. via resonance in the vibronic region of the 1250 nm absorption band of this species. This spectrum indicates that the unpaired electron in dimeric P +. is not equally shared between the two BChl molecules constituting the primary donor. Spectral contributions of the carotenoid in the FT RR spectra of wild-type Rb. sphaeroides (2.4.1) RCs confirm it is assuming an out-of-plane distorted, 15- cis configuration under the specific conditions offered by FT Raman spectroscopy, i.e. with the sample at room temperature and using an excitation wavelength which is non-actinic for the carotenoid.

A structural role of the carotenoid in the light-harvesting II protein of Rhodobacter capsulatus

The membrane-linked light-harvesting II protein (LHII) of Rhodobacter capsulatus was partly depleted of carotenoids by selective extraction with light petroleum. Carotenoid removal was accompanied by bleaching of the Qy(S1<--S0) absorption band of bacteriochlorophyll (Bchl) a near 800 nm, by a bathochromic shift and a broadening of the other Bchl Qy band at 850 nm, and by the formation of a weak Qy band of dissociated Bchl near 770 nm. The changes in the 800 and 850 nm bands seemed to reflect alterations in only those Bchl molecules that had lost their associated carotenoids, firstly, because the extent of the changes was closely correlated to the degree of carotenoid extraction, and, secondly, because the residual fraction of carotenoid-containing LHII, which could be almost quantitatively recovered from the membrane after detergent solubilization and ion-exchange chromatography, showed an unmodified LHII absorption spectrum. The Bchl responsible for the shifted 850 nm band remained bound to protein, since its visible (Qx) transition seemed to retain the induced optical activity of the native bound pigment. Besides, the shifted Bchl could act as an efficient acceptor of singlet excitation energy from the pigments of the intact LHII fraction. The close similarity between the spectroscopic Bchl changes that accompany carotenoid extraction and the differential spectral features of carotenoidless LHII of Rhodobacter mutants, previously reported, strongly suggests that the direct cause of the spectral modifications is the absence of carotenoid and not any independent effect of the experimental manipulation of the membrane. Several interpretations of the structural changes that underlie the observed spectral changes are possible. The simplest one is to assume that carotenoid removal elicits an alteration in the angle between the Qy transition moments of two strongly interacting Bchl molecules.

SPECTRAL MODIFICATIONS OF BACTERIAL ANTENNA COMPLEXES BY LIMITED PROTEOLYSIS

AbstractThe spectral effects of a number of endo‐ and exoproteases to the detergent‐solubilized antenna complexes B802–858(sphaeroides like) of strain Rhodopseudomonas acidophila 10050 and B802–824of strain 7050 were analyzed. Upon protease digestion the time course of spectral modification between 250 and 900 nm was recorded. In addition, circular dichroism (CD) signals were measured between 450 and 950 nm. The endoprotease elastase as well as the exoprotease carboxypeptidase A (CP A) altered the B802–858antenna complex, by changing the 858 nm band hypochromically (30‐50%) with an additional small hypsochromic shift (3–6nm). A combined carboxypeptidase digestion, e.g. CP A and CP B, yielded a further modification to a complex with absorption bands at 800 nm and ˜840 nm (ε˜ 50% of the 800 nm band). By exposing the isolated B803–824antenna complex to the different proteases no significant spectral change was observed. The B802–858antenna complex, modified by CP A, exhibits a60–70% decrease of the biphasic CD signal in the near infrared. The limited proteolysis experiments provide conclusive experimental evidence that the C‐terminal domains of the antenna polypeptides α and β contribute to the formation of (dimeric) bacteriochlorophyll (BChl) a molecules absorbing at around 850 nm. This agrees with the hypothesis, based on comparative amino acid sequence analysis, that in purple bacterial antenna complexes the structural requirements (specific amino acids in the vicinity of the BChl molecules, e.g. aromatic amino acids) for batho‐ and hyperchromicity of BChl molecules apparently reside to a considerable extent in the C‐terminal portions of their antenna apoproteins.

Ultrafast dynamics of excitation energy transfer and trapping in Bchl a and Bchl b-containing photosynthetic bacteria

Using IR transient absorption spectroscopy we have studied how energy migrates through the light-harvesting antenna of photosynthetic purple bacteria, and how the energy is trapped by the reaction centre. In bacteriochlorophyll (Bchl) a-containing purple bacteria the light-harvesting antenna is highly heterogeneous, consisting of several spectroscopically distinct pigments. Energy transfer between these pigments occurs on several different time scales. The overall trapping time at room temperature is about 60 ps, while energy transfer from the high-energy to the low-energy pigments of the light-harvesting antenna only takes approximately 10 ps (measured at 77 K). This implies that trapping is relatively slow, approximately 35 ps at 77 K, and probably not much faster at room temperature. Energy transfer between neighbouring Bchl molecules within a pigment—protein complex occurs on the 1 ps to subpicosecond time scale. For the Bchl b-containing purple bacterium Rhodopseudomonas viridis the results show that the antenna appears homogeneous at room temperature and 77 K, and that trapping appears to be very fast, and approximately described by a random-walk model using known spectral properties of the antenna and reaction centre also appear to be much more tightly coupled than in the Bchl a-containing purple bacteria.

Antenna organization of Rhodopseudomonas acidophila: a study of the excitation migration

Transfer of excitation energy was studied in three different cultures of the purple bacterium Rhodopseudomonas acidophila , which all contained the core antenna complex B880, but differed with respect to the type of peripheral antenna complex, which was either B800–820, B800–850, or both. Contrary to that of B800–850, the fluorescence emitted by B800–820 did not increase upon light-induced oxidation of P-870. Singlet-singlet annihilation measurements showed that in cells containing only B880 and B800–820 energy transfer can take place within a domain of 50 ± 20 bacteriochlorophyll (BChl) molecules, which number equals that of BChls per reaction center. In cells containing B800–850 energy transfer can take place over at least 200 BChls of B880. Our results are explained by a model where small clusters of B880, the size of one photosynthetic unit, are separated from each other by a more or less continuous array of B800–820 or B800–850 complexes. B800–820, but not B800–850, acts as an energy barrier between different B880 units.