No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
The effect of chemical oxidation on the fluorescence of the LH1 (B880) complex from the purple bacterium Rhodobium marimum
Abstract
The effect of chemical oxidation on the absorption and fluorescence emission spectra of the LH1 complex from Rhodobium marinum was investigated. Mild chemical oxidation of the LH1 complex, by addition of 10 mM potassium ferricyanide, caused a 2-3% bleaching of the 880-nm Qy absorption band. In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the LH1 complex was quenched by about 50%. This result demonstrates that oxidation of very few bacteriochlorophyll (BChl) molecules in the LH1 ring is enough to completely quench its fluorescence.
... This effect was later confirmed for action of an oxidant on isolated complexes [20]. A study of LH1 com plex from Rhodobium marinum showed that even minor BChl880 oxidation under low potassium ferricyanide concentration (10 mM) causes significant fluorescence quenching for this BChl [21]. Using EPR and electron nuclear double resonance (ENDOR), the formation of a BChl cation radical was found in LH1 samples [22 24]. ...
... minutissimum mem branes is irreversible, and the intensity of the absorption bands cannot be restored even partially after removal of the oxidant by gel filtration (Fig. 1, spectrum 3). A simi lar conclusion about the irreversibility of such changes was reported in the literature [20,21,26]. A differential spectrum ( Fig. 1, spectrum 4) confirms the main target of potassium ferricyanide to be BChl850 of LH2 complex and, partially, BChl890 of LH1 complex. ...
... Fluorescence intensity in oxidized LH2 complex is approximately five times lower than in a control sample. Even a minor long wave length BChl oxidation in LH1 RC complex is known to cause a significant quenching of its fluorescence [21]. It could be expected that BChl800 fluorescence (815 825 nm range) would appear in the fluorescence spectrum Fig. 3. a) Absorption spectrum of LH2 complex from Alc. minutissimum before (1) and after treatment with 100 mM potas sium ferricyanide with subsequent removal of the oxidant by gel filtration (2). ...
Oxidation of bacteriochlorophyll (BChl) with potassium ferricyanide in membranes and LH2 complexes (carotenoid-less and control samples) from the purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides as well as BChl photobleaching in a model system have been studied. The oxidation of BChl depended on the type of bacteria. BChl850 was rapidly oxidized in samples from Alc. minutissimum, and BChl800 and BChl850 were slowly oxidized in samples from Rb. sphaeroides. The carotenoids were not involved in protecting BChl from chemical oxidation in the lightharvesting complexes. The appearance of BChl oxidation product was registered in the absorption spectra (absorption maximum about 700 nm) and by HPLC analysis. The oxidized BChl was identified as 3-acetyl-chlorophyll. It differed from BChl by the presence of a double bond in pyrrole ring II at the 7-8 position. The extinction coefficient of 3-acetyl-chlorophyll was about 10 times less than that of BChl850 in the LH2 complex from Alc. minutissimum. In the BChl → 3-acetylchlorophyll transition, the binding constant of the latter with LH2 complex as compared with that of BChl did not change dramatically, as indicated by: (i) preserved electrophoretic mobility of the complex; (ii) the presence of 3-acetyl-chlorophyll in the complex after separation; (iii) the presence of a 3-acetyl-chlorophyll CD signal that was proportional to its absorption spectrum.
... It has been shown that BChl components of LH1 complexes undergo oxidation upon treatment with potassium ferricyanide. Such treatment leads to changes in the absorption, circular dichroism (CD), fluorescence emission, and electron paramagnetic resonance spectral signals of the complex (Gomez et al., 1982;Picorel et al., 1984;Law et al., 1998). We have previously studied the effects of Crts on stability of BChla and its derivatives in a model system (Fiedor et al., 2001a;Fiedor et al., 2002), however, so far, the effect of Crts on stability of LH1 complexes exposed to chemical oxidation has not been demonstrated. ...
... 2-desvinyl-2-acetylchlorophyll a, as judged from the appearance of a band at ~690 nm in the absorption spectra (Smith et al., 1966). Picorel et al. (1984) or Law & Cogdell (1998) have already demonstrated that oxidation of native LH1 complexes with K 3 Fe(CN) 6 leads to the changes in their spectral properties. Similar changes in the absorption and emission spectra of the reconstituted Sph-LH1 complexes were seen in the present work. ...
... The changes in the absorption spectra of LH1 caused by oxidants are slower than the ones in the emission signals. A more rapid drop in the emission intensity can be explained by the fact that the presence of even a small number of excitation traps (oxidized BChl) may completely inhibit the intracomplex energy transfer (Law et al., 1998;Fiedor et al., 2000;Fiedor et al., 2001b). ...
The effect of carotenoids on stability of model photosynthetic pigment-protein complexes subjected to chemical oxidation with hydrogen peroxide or potassium ferricyanide was investigated. The oxidation of carotenoid-less and carotenoid-containing complexes was conducted in the presence or absence of ascorbic acid. The progress of the reactions was monitored by use of absorption and fluorescence spectroscopy. Our results show that carotenoids may significantly enhance the stability of photosynthetic complexes against oxidation and their protective (antioxidant) effect depends on the type of the oxidant.
... In all photosynthetically active chlorophyll proteins, they are accompanied by carotenoids that, among other functions, accept and quench excess excitation energy from the chlorophylls [13]. There is evidence that under certain circumstances Chls can also degrade excess excitation energy in a yet poorly understood fashion [14,15]. In all chlorophyll-protein proteins, the pigments also contribute to the stabilization of the photosynthetic complexes [16][17][18][19][20]. ...
... One example is cation radicals. They have been generated artificially in purple bacterial LH complexes, but may also arise in vivo under saturating light conditions [14]. Another example are chlorosomes: in model aggregates devoid of carotenoids, a conformational(?) change can be induced by intense light pulses, which leads to excited state quenching [15]. ...
... In contrast to the exchange of B800 BChl a, there is little information on in situ conversion of BChl a into Chl-type pigments in LH2 proteins. Especially, no information is available about the isolation and the detailed characterization of LH2 proteins whose B800 BChl a is selectively converted to Chl pigments by in situ oxidation, although BChl oxidation has been reported in studies on photoprotection in light-harvesting proteins in purple bacteria [18][19][20][21][22][23][24] and the functional roles of BChl pigments in proteins in green sulfur bacteria 25 and heliobacteria 26 . Generally bacteriochlorin pigments (7,8,17,18-tetrahydroporphyrins) are chemically oxidized by 2,3-dichloro-5,6 -dicyano-1,4-benzoquinone (DDQ) to give corresponding chlorins (17,18-dihydroporphyrins) 27 . ...
... These results are in sharp contrast to our results, which clearly indicate that only B800 BChl a was exclusively converted to 3-acetyl Chl a. The differences are mainly ascribable to the oxidants used for the chemical oxidation of LH2 proteins; ferricyanide was commonly used in previous studies 18,[21][22][23]25 . Ferricyanide produces a cation radical of BChl a, which is subsequently converted to oxidized products, since ferricyanide acts as a one-electron oxidant. ...
Engineering chlorophyll (Chl) pigments that are bound to photosynthetic light-harvesting proteins is one promising strategy to regulate spectral coverage for photon capture and to improve the photosynthetic efficiency of these proteins. Conversion from the bacteriochlorophyll (BChl) skeleton (7,8,17,18-tetrahydroporphyrin) to the Chl skeleton (17,18-dihydroporphyrin) produces the most drastic change of the spectral range of absorption by light-harvesting proteins. We demonstrated in situ selective oxidation of B800 BChl a in light-harvesting protein LH2 from a purple bacterium Rhodoblastus acidophilus by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The newly formed pigment, 3-acetyl Chl a, interacted with the LH2 polypeptides in the same manner as native B800. B850 BChl a was not oxidized in this reaction. CD spectroscopy indicated that the B850 orientation and the content of the α-helices were unchanged by the B800 oxidation. The nonameric circular arrangement of the oxidized LH2 protein was visualized by AFM; its diameter was almost the same as that of native LH2. The in situ oxidation of B800 BChl a in LH2 protein with no structural change will be useful not only for manipulation of the photofunctional properties of photosynthetic pigment-protein complexes but also for understanding the substitution of BChl to Chl pigments in the evolution from bacterial to oxygenic photosynthesis.
... This may be a reason for the low oxygen tolerance of this species (Laußermair and Oesterhelt, 1992). Triplets can also be produced in antenna complexes, where the mechanism is unclear but may also involve radical recombination reactions (Law and Cogdell, 1998) Last but not least, carotenoids are quenchers of reactive oxygen species ( 1 O 2 , OH -) in case the aforementioned first defense lines become saturated or inefficient. Since the reaction 5 ANT002 Chapter 2 The Pigments ...
... Biliprotein triplets have been implicated in the generation of reactive oxygen species by biliproteins (He et al., 1996), but to the best of this author's knowledge they have never been positively identified, and several checks by ODMR have failed (unpublished). Chl cation radicals, which can be generated in antennas under saturating conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). ...
The Pigments of antenna stystems have several functions. 1: they absorb light efficiently, 2: they transfer the excitation energy with minimum losses to the reaction centers, 3: They degrade excess energy to heat, and 4: they contribute to the stabilization and regulation of the photosynthetic apparatus. The pigments used to fulfill these functions are chlorophylls, phycobilins and carotenoids. This chapter gives the structures, biosynthetic pathways and spectroscopic properties for each pigment type. Examples are given of strategies to study exctitation energy transfer by selective introduction of specifically modified chromophores.
... This may be a reason for the low oxygen tolerance of this species (Laußermair and Oesterhelt, 1992). Triplets can also be produced in antenna complexes, where the mechanism is unclear but may also involve radical recombination reactions (Law and Cogdell, 1998) Last but not least, carotenoids are quenchers of reactive oxygen species ( 1 O 2 , OH -) in case the aforementioned first defense lines become saturated or inefficient. Since the reaction 5 ANT002 Chapter 2 The Pigments ...
... Biliprotein triplets have been implicated in the generation of reactive oxygen species by biliproteins (He et al., 1996), but to the best of this author's knowledge they have never been positively identified, and several checks by ODMR have failed (unpublished). Chl cation radicals, which can be generated in antennas under saturating conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). ...
Chlorophylls and carotenoids are essential pigments of photosynthesis, in which they have complementary functions for capturing light and transducing it to biochemical energy and for protecting against its deleterious effects. Carotenoids are also present in most other organisms where they have, besides photoprotection, a variety of other functions. Typical structures of both types of pigments are reviewed in this article as well as their functions, chemical properties, spectroscopy, biosynthesis, and applications.
... The observation of subsequent bleaching of the two Chl a molecules in reconstituted PCP complexes, together with the insensitivity of the fluorescence emission of the second Chl to the bleaching of the first one, demonstrates weak dipole-dipole coupling between the Chls within a PCP monomer. First of all, it has been shown that oxidized BChl molecules are very efficient quenchers of the fluorescence in LH1 [24] and LH2 [16] complexes. Controlled chemical oxidation, which induces only slight (∼ 2%) changes in the absorption of LH1, leads already to 50% reduction of the fluorescence [24]. ...
... First of all, it has been shown that oxidized BChl molecules are very efficient quenchers of the fluorescence in LH1 [24] and LH2 [16] complexes. Controlled chemical oxidation, which induces only slight (∼ 2%) changes in the absorption of LH1, leads already to 50% reduction of the fluorescence [24]. Strongly interacting Chl a molecules should also exhibit very similar behavior: upon bleaching of one Chl molecule, the fluorescence intensity of the second Chl should be dramatically reduced. ...
Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.
... Earlier, a BChl +• monocation radical that absorbs at around 900 nm was proposed as a potential fluorescence quencher in bacterial antennas. 8 The presence of this one-electron oxidation product in our samples cannot be excluded. However, its amount should be rather small. ...
As a basis of photosynthesis, photo-induced oxidation of (bacterio)chlorophyll molecules in the special reaction center complexes has been a subject of extensive research. In contrast, the generally harmful photooxidation of antenna chromoproteins has received much less attention. Here, we have established the permanent structural changes in the LH2 antenna bacteriochlorophyll-protein complex from a sulfur photosynthetic purple bacterium Ectothiorhodospira haloalkaliphila taking place at physiological conditions upon intense optical irradiation. To this end, a crystal structure of the LH2 complex from Ectothiorhodospira haloalkaliphila was first resolved by X-ray diffraction to 3.6 Å, verifying a great similarity with the earlier structure from Phaesporillum molischianum. Analysis of the various steady-state and picosecond time-resolved optical spectroscopy data and related model simulations then confirmed that the major spectral effects observed – bleaching and blue-shifting of the B850 exciton band, and correlated emergence of a higher-energy C700 exciton band – are associated with photooxidation of increasing numbers of B850 bacteriochlorophylls into 3-acetyl-chlorophylls with no noticeable damage to the pigment-binding protein scaffold. A prospective non-invasive method for an in situ optical control of excitons by selective photooxidation of pigment chromophores was thus revealed and demonstrated in a structurally well-defined native system.
... Competitive titration with potassium ferricyanide Potassium ferricyanide [K 3 Fe(CN) 6 ] is a known fluorescence quencher (Law and Cogdell 1998). To find out the origin of the enhancement of fluorescence intensity during the titration of C-GNPs and DM, studies were carried out with C-GNPs and varying concentration of [K 3 Fe(CN) 6 ] at two different conditions. ...
A colorimetric chemo-sensor based on citric acid-coated gold NPs (C-GNP) showed a linear increase in fluorescence intensity with increasing concentration of pesticide dimethoate (DM). The limit of detection was found to be between ~8.25± 0.3 and 20 ± 9.5 ppm. The increase in fluorescence intensity was suggested to have originated from the soft–soft interaction between C-GNPs and DM via sulfur group which is absent in pesticide dicofol (DF). Similar studies with citric acid-coated silver NPs (C-SNPs) did not result any change in the fluorescence intensity. The microscopic studies suggested aggregation of C-GNPs in the presence of DM but not in case of DF.
Graphical Abstract
... Carotenoid-less mutants are highly light-sensitie in all photosynthetic organisms and are unable to surie in the natural enironment. Comparatiely little is known about the protectie role, if any, of Chls: Chl cation radicals, which can be generated in antennas under saturating light conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). Most chlorophyll aggregates are also excellent quenchers (Katz et al., 1978b;Scherz et al., 1991), with the notable exception of those of BChls c, d and e in the chlorosomes (Blankenship and Matsuura, 2003; Chapter 20, deBoer and deGroot). ...
... As before the amplitude of the 600 ps component does not change significantly as a function of the K 3 Fe(CN)6 concentration. We note that we observe a strong quenching of the RC-LH1 fluorescence as well as a bleaching of the B875 absorption band for K 3 Fe(CN)6 concentrations above 100 µM, which is likely to be caused by an oxidation and concomitant destruction of the LH1 ring as has been found before[120].Apparently, the increase of the concentration of the reducing agent, i.e. shifting the steady state population of the RCs to the neutral state P (RC open), has qualitatively the same effect (increase of the 40 ps component; decrease of the 200 ps component) as decreasing the excitation rate for an unmanipulated sample. Vice versa, increasing the concentration of the oxidant, i.e. shifting the steady-state populations of the special pairs towards the P+ state (RC closed) has the same effect (decrease of the 40 ps component; increase of the 200 ps component) as increasing the excitation rate for an unmanipulated sample. ...
Multichromophoric systems play a major role in natural photosynthetic processes. They are the basic building blocks for the absorption of light and the subsequent energy transfer within photosynthetic organisms. Optimized by evolutionary selection processes, they constitute an ideal blueprint for artificial, nature-inspired multichromophoric light-harvesting systems. This thesis investigates the energy transfer and the photophysical properties of different multichromophoric systems:
-Reaction centre light-harvesting complex I (RC-LH1), a natural light-harvesting complex from purple bacteria,
-a hybrid system built from a spherical gold nanoparticle (AuNP) and a light-harvesting complex II (LH2), another light-harvesting complex from purple bacteria,
-two novel artificial light-harvesting systems, based on carbonyl-bridged triarylamines (CBTs)
Different methods of optical spectroscopy and optical microscopy have been used to investigate these systems.
The first part of this work discusses the results of time-resolved optical spectroscopy in a picosecond-range on isolated RC-LH1 from Rhodopseudomonas palustris. Their fluorescence decay was recorded dependent on the excitation fluence and the repetition rate of the excitation laser. Both parameters were varied over three orders of magnitude. We observed three components in the decays with characteristic decay times of 40, 200 and 600 ps, respectively, that occurred in different amplitude ratios, dependent on the excitation parameters. A first suggestion that the decay times are an indicator of the redox state of the so called “special pair” (P) within the RC could be underpinned by two reference experiments. In those experiments, the redox state of P was influenced by a reducing agent or an oxidising agent, respectively, by stepwise increasing its concentration while the fluence and the repetition rate stayed fixed. Thus the decay times of 40, 200 and 600 ps could be assigned to different species of RC-LH1 with their special pair in the neutral state P, with the special pair in the reduced state P+ and with the reaction centre lacking or dysfunctional, respectively. Using these results as well as values from the literature we were able to design a detailed kinetic model of the energy transfer pathways in RC-LH1. The fluorescence decays of RC-LH1 could then be simulated by a global master equation approach based on a microstate description. Due to an excellent agreement between experiment and simulation this model allows to predict the relative populations of the aforementioned species for given excitation parameters as well as to predict the relative population of carotenoid triplet states on the LH1 rings.
In the second part of this work, a fluorescence microscope with single molecule sensitivity was used to show the plasmonic fluorescence enhancement of LH2 from Rhodobacter sphaeroides by spherical AuNP. The fluorescence intensities of single LH2 were measured in presence and in absence of AuNP, with the excitation wavelength in resonance and off resonance of the AuNP’s plasmon. Using a home-built evaluation algorithm the intensities of more than 4000 single LH2 could be retrieved. From these intensities, extensive distributions that show the relative frequencies of the different intensity values for all the aforementioned excitation conditions could be gained. When excited in resonance with the plasmon of the AuNP, the intensity distribution of the LH2 in presence of AuNPs as a whole shifted to higher intensity values as compared to the case in absence of AuNPs. The mean value of the intensity distribution in presence of the AuNP was about a factor of 2 higher than that of the intensity distribution in absence of the AuNP. When excited off the plasmon resonance, the intensity distributions of LH2 were almost identical in presence as well as in absence of AuNPs. These observations show the plasmonic origin of the fluorescence enhancement of LH2 and point towards a small spatial distance between LH2 and AuNP, which might indicate an adsorption of LH2 to the spherical AuNP. In a reference experiment a spacer layer of a thickness of 20 nm was introduced between AuNP and LH2. In this configuration, no fluorescence enhancement could be detected even at excitation of LH2 within the plasmon resonance. The result from this reference experiment thus defined a maximum distance between AuNP and LH2 of 20 nm when no spacer layer is present. Model calculations underpinned our observations and gave a direct hint towards an adsorption of LH2 to the AuNPs.
The third part of this work is concerned with the extensive optical characterisation of two novel organic light-harvesting systems. Two Sprache: Englisch
Multichromophoric systems play a major role in natural photosynthetic processes. They are the basic building blocks for the absorption of light and the subsequent energy transfer within photosynthetic organisms. Optimized by evolutionary selection processes, they constitute an ideal blueprint for artificial, nature-inspired multichromophoric light-harvesting systems. This thesis investigates the energy transfer and the photophysical properties of different multichromophoric systems:
-Reaction centre light-harvesting complex I (RC-LH1), a natural light-harvesting complex from purple bacteria,
-a hybrid system built from a spherical gold nanoparticle (AuNP) and a light-harvesting complex II (LH2), another light-harvesting complex from purple bacteria,
-two novel artificial light-harvesting systems, based on carbonyl-bridged triarylamines (CBTs)
Different methods of optical spectroscopy and optical microscopy have been used to investigate these systems.
The first part of this work discusses the results of time-resolved optical spectroscopy in a picosecond-range on isolated RC-LH1 from Rhodopseudomonas palustris. Their fluorescence decay was recorded dependent on the excitation fluence and the repetition rate of the excitation laser. Both parameters were varied over three orders of magnitude. We observed three components in the decays with characteristic decay times of 40, 200 and 600 ps, respectively, that occurred in different amplitude ratios, dependent on the excitation parameters. A first suggestion that the decay times are an indicator of the redox state of the so called “special pair” (P) within the RC could be underpinned by two reference experiments. In those experiments, the redox state of P was influenced by a reducing agent or an oxidising agent, respectively, by stepwise increasing its concentration while the fluence and the repetition rate stayed fixed. Thus the decay times of 40, 200 and 600 ps could be assigned to different species of RC-LH1 with their special pair in the neutral state P, with the special pair in the reduced state P+ and with the reaction centre lacking or dysfunctional, respectively. Using these results as well as values from the literature we were able to design a detailed kinetic model of the energy transfer pathways in RC-LH1. The fluorescence decays of RC-LH1 could then be simulated by a global master equation approach based on a microstate description. Due to an excellent agreement between experiment and simulation this model allows to predict the relative populations of the aforementioned species for given excitation parameters as well as to predict the relative population of carotenoid triplet states on the LH1 rings.
In the second part of this work, a fluorescence microscope with single molecule sensitivity was used to show the plasmonic fluorescence enhancement of LH2 from Rhodobacter sphaeroides by spherical AuNP. The fluorescence intensities of single LH2 were measured in presence and in absence of AuNP, with the excitation wavelength in resonance and off resonance of the AuNP’s plasmon. Using a home-built evaluation algorithm the intensities of more than 4000 single LH2 could be retrieved. From these intensities, extensive distributions that show the relative frequencies of the different intensity values for all the aforementioned excitation conditions could be gained. When excited in resonance with the plasmon of the AuNP, the intensity distribution of the LH2 in presence of AuNPs as a whole shifted to higher intensity values as compared to the case in absence of AuNPs. The mean value of the intensity distribution in presence of the AuNP was about a factor of 2 higher than that of the intensity distribution in absence of the AuNP. When excited off the plasmon resonance, the intensity distributions of LH2 were almost identical in presence as well as in absence of AuNPs. These observations show the plasmonic origin of the fluorescence enhancement of LH2 and point towards a small spatial distance between LH2 and AuNP, which might indicate an adsorption of LH2 to the spherical AuNP. In a reference experiment a spacer layer of a thickness of 20 nm was introduced between AuNP and LH2. In this configuration, no fluorescence enhancement could be detected even at excitation of LH2 within the plasmon resonance. The result from this reference experiment thus defined a maximum distance between AuNP and LH2 of 20 nm when no spacer layer is present. Model calculations underpinned our observations and gave a direct hint towards an adsorption of LH2 to the AuNPs.
The third part of this work is concerned with the extensive optical characterisation of two novel organic light-harvesting systems. Two derivatives of carbonyl-bridged triarylamines (CBTs) were investigated with absorption spectroscopy, photoluminescence (PL) emission and PL-excitation spectroscopy (all steady-state) as well as time-resolved emission spectroscopy on picosecond timescales. Both compounds consist of a CBT core that is decorated with either three peripheral naphthalimide (NI) molecules or three peripheral 4-(5-hexyl-2,2’-bithiophene)-naphthalimide (NIBT) molecules. These compounds are abbreviated as CBT-NI and CBT-NIBT, respectively. Additionally isolated CBT, NI and NIBT were investigated as reference compounds as well as mixtures of CBT with NI or NIBT, respectively, that were not covalently bound. For all compounds, we recorded the absorption spectra from the near ultraviolet to the near infrared range, the PL emission spectra in dependence of the excitation wavelength – which at the same time gave access to the PL-excitation spectra – as well as the PL quantum yield and the PL lifetime. For the mixtures of the isolated compounds the same parameters were recorded except for the PL quantum yield and the PL lifetime. The data showed that CBT-NI works as an energy funnel. Photoluminescence always occurred from the CBT core, no matter whether the excitation was tuned to the spectral region of the CBT core or the peripheral NI. For CBT-NIBT, the reverse behaviour could be observed. No matter which absorption band was excited, only PL from the NIBT periphery could be observed. In contrast to the concentrator abilities of CBT-NI, CBT-NIBT represents an energy distributor.
In summary this thesis presented three multichromophoric systems that act as examples to understand natural light-harvesting systems, to manipulate them and to imitate them. The experiments on RC-LH1 from Rhodopseudomonas palustris allow a deeper understanding of the energy transfer pathways within this system. The model derived from these experiments enables us to make detailed predictions of the photophysical behaviour of RC-LH1 dependent on the excitation parameters. The study on AuNP-LH2 hybrids represents a statistically solid proof-of-principle that shows the plasmonic fluorescence enhancement of single bacterial light-harvesting complexes by well-defined nanostructures. It gives a good example on how natural light-harvesting systems can be manipulated. The imitation and advancement of natural light-harvesting concepts is realised in the study of the two derivatives of CBT. The extensive optical characterisation of these novel compounds shows their potential as building blocks for molecular electronics, organic photovoltaics and as a promising model system for molecular energy transfer.
... Carotenoid-less mutants are highly light-sensitie in all photosynthetic organisms and are unable to surie in the natural enironment. Comparatiely little is known about the protectie role, if any, of Chls: Chl cation radicals, which can be generated in antennas under saturating light conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). Most chlorophyll aggregates are also excellent quenchers (Katz et al., 1978b;Scherz et al., 1991), with the notable exception of those of BChls c, d and e in the chlorosomes (Blankenship and Matsuura, 2003; Chapter 20, deBoer and deGroot). ...
... Why are the LH1 Bchla molecules not positioned even closer to the P870 in this case ? If even one molecule of Bchla in the LH1 ' ring ' is oxidized inadvertently, the fluorescence yield (which is equivalent to the lifetime of the singlet excited state) is very strongly quenched (Law & Cogdell, 1998). This would then prevent LH1 from acting as an effective antenna for the RC. ...
This review sets out the case that now is the time for plant science to establish the technologies required for routinely studying the structure and function of plant proteins. The impact that protein structural information can have is illustrated here with reference to photosynthesis. Our understanding of the precise molecular mechanisms of the light‐reactions of photosynthesis has been transformed by the combination of high‐resolution protein structural data and detailed functional studies. The past few years have been a particularly exciting time to be engaged in basic plant science research. The application of modern techniques of molecular biology has allowed many key questions to be addressed. The stage is now set for an even bigger revolution as the current plant genome sequencing projects are completed. If these advances are going to be fully exploited, plant science must get to grips with studying proteins, not just genes. Reliable methods for the overexpression of proteins in their native state coupled with routine access to structure determination must become the norm rather than the exception. In 1998 there were about 9000 protein structures deposited in the Brookhaven database. Very few of these are plant proteins. This trend will have to be reversed if research in molecular plant science is to fulfil its potential.
contents
Summary
167
I.
introduction
168
II.
the impact of protein structural information on the understanding of the primary reactions in photosynthesis
169
III.
developing overexpression systems for the study of plant protein
188
IV.
conclusions
190
... Carotenoid-less mutants are highly light-sensitie in all photosynthetic organisms and are unable to surie in the natural enironment. Comparatiely little is known about the protectie role, if any, of Chls: Chl cation radicals, which can be generated in antennas under saturating light conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). Most chlorophyll aggregates are also excellent quenchers (Katz et al., 1978b;Scherz et al., 1991), with the notable exception of those of BChls c, d and e in the chlorosomes (Blankenship and Matsuura, 2003; Chapter 20, deBoer and deGroot). ...
The chlorophylls are a structurally and functionally distinct group of macrocyclic tetrapyrrole pigments that may occur at
the porphyrin, chlorin or bacteriochlorin oxidation levels. Biosynthetically, they are derived from protoporphyrin IX. Structurally,
they are characterized by the presence of a fifth ring, isocyclic ring E, which confers the prefix ‘phyto’ to the porphyrin
and chlorin type molecules. Chlorophylls generally have Mg as the central metal and a long-chain esterifying alcohol at C-173.
... These observations can be rationalized by a two-component system, comprising only (i) a subpopulation of complexes with native fl uorescence quantum yield and a decay-time of about 850 ps, and (ii) a second sub-population of complexes, which are practically non-fl uorescent on the time-scale of several tens of picoseconds. A very similar effect on the fl uorescence was observed when another type of excitation trap, a chemically induced BChl a cation radical, was introduced into LH1 from Rhodobium marinum (Law and Cogdell, 1998). The pigment unit size in reconstituted LH1 sub-stituted with increasing amounts of Ni-BChl a can be determined via statistical analysis of the emission quenching data. ...
Two complementary model systems are described, which are used to study the assembly of functional light-harvesting (LH) complexes.
One system is based on rational design of cofactor-binding motifs and their capacity to assembly model LH2 complexes via expression
in native-like membranes. The second takes advantage of the highly reversible self-assembly of the LH1 complex in artificial
membranes and provides a convenient tool for design of model complexes with modified cofactors. In essence, re-design of the
cofactor binding pockets in LH2 enables exploration of the underlying principles that enable particular amino acid combinations
to sustain stable and functional assembly of LH-active arrays. Cofactor-binding motifs predicted in silico are tested in the context of the LH2 complex. In this way, H-bonding at the bacteriochlorophyll (BChl)/protein interface
and the presence of aromatic residues were identified as critical for assembly of BChl and carotenoid (Crt). Moreover, the
volumes of particular residues in the vicinity of BChl were shown to be critical for fine-tuning the spectroscopic properties.
The LH1 reconstitution system, on the other hand, provides new information on the cofactor-related determinants of formation
and functioning of this LH complex. Using the excitation trap approach, the coupling between BChl and excitation delocalization
over the LH1 ring could be evaluated, while, by the replacement of Crts, their contribution to the assembly was assessed and
for the first time a Crt-binding intermediate of LH1 assembly was identified. A new challenge is to make the two model approaches
more interchangeable, thus allowing us to compare the same factors in different LH complexes, and eventually to identify on
a molecular level what renders these apparently similar complexes so different.
... Carotenoid-less mutants are highly light-sensitie in all photosynthetic organisms and are unable to surie in the natural enironment. Comparatiely little is known about the protectie role, if any, of Chls: Chl cation radicals, which can be generated in antennas under saturating light conditions, are good quenchers of Chl excited states (Law and Cogdell, 1998). Most chlorophyll aggregates are also excellent quenchers (Katz et al., 1978b;Scherz et al., 1991), with the notable exception of those of BChls c, d and e in the chlorosomes (Blankenship and Matsuura, 2003; Chapter 20, deBoer and deGroot). ...
Supplementary material to this book contains the following Adobe-Writer (.pdf) files: an overview of the material, the color coding for the map on the title page), and supporting information for chapter 1, 14, 20, 22 and 30. Some of the files contain further links to materials on this server, in particular is there a collection of structural formulas accessible from the material to chapter 1.
Please, click on the respective files for downloading. Any reference should cite the full title of the book.
... Under steady illumination the B850 fluorescence suddenly would be extinguished, which was attributed to the photoformation of an excitation trap, proposed to be a radical cation, in the B850 ring. Indeed oxidation of about 1 Bchl molecule per ring in the structurally homologous LH1 ring results in complete quenching of its fluorescence (14). Continued illumination often led to the recovery of the fluorescence. ...
Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.
... The LH1 complex was prepared as described previously (Law and Cogdell 1998). ...
The reaction center (RC) and the core (RC-LH1) complex were isolated and purified from Rhodobium marinum; together with the LH1 complex [Meckenstock et al. (1992a) FEBS Lett. 311: 128], a complete set of RC, LH1 and RC-LH1 from the same wild-type strain of a purple photosynthetic bacterium can therefore now be made. Comparison of the BChl a/BPhe a ratio (determined by HPLC) between the RC and the RC-LH1 complexes lead us to the determination of the number of BChls in the LH1 ring to be 32.06+/-2.90, indicating that the LH1 ring from Rh. marinum consists of 16 alphabeta subunits.
... Rh. marinum DSM 2698 was cultured anaerobically at 30ЊC with a light intensity of ϳ3400 lx in the DSM-27 Rhodospirillaceae medium supplemented with 3% NaCl (11). The LH1 was prepared as described previously (13). The preparations of RC and RC-LH1 have been described elsewhere (12). ...
Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and 1H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time-dependent changes in the carotenoid composition in the reaction center (RC) and the light-harvesting complex 1 (LH1) were traced by high-performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.
... Bchl a molecules not positioned nearer to the RC special pair Bchls? Oxidation of just a single Bchl a molecule in LH1 results in a strong quenching of the fluorescence yield (which is equivalent to the singlet excited state lifetime) thereby preventing LH1 from acting as an effective antenna for the RC (Law and Cogdell 1998 ...
The harvesting of solar radiation by purple photosynthetic bacteria is achieved by circular, integral membrane pigment-protein complexes. There are two main types of light-harvesting complex, termed LH2 and LH1, that function to absorb light energy and to transfer that energy rapidly and efficiently to the photochemical reaction centres where it is trapped. This mini-review describes our present understanding of the structure and function of the purple bacterial light-harvesting complexes.
... However, by manipulating and exploiting the optically forbidden but longer lived (picoseconds) S 1 excited state of the carotenoids, the LH complexes of PCP improve the carotenoid to Chl EET yield to almost 100% [76]. Cation and anion radicals are also very efficient quenchers of excitation energy [77]. The proximity of LH and RC pigments required for efficient EET increases the risk of forming such radicals by short-circuiting the energy and electron transfer units. ...
Decades of research on the physical processes and chemical reaction-pathways in photosynthetic enzymes have resulted in an extensive database of kinetic information. Recently, this database has been augmented by a variety of high and medium resolution crystal structures of key photosynthetic enzymes that now include the two photosystems (PSI and PSII) of oxygenic photosynthetic organisms. Here, we examine the currently available structural and functional information from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in de novo designed and custom-built molecular solar energy conversion devices. We find that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Moreover, we find that the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. Thus, for projected de novo designed constructions, the control of spatial organization of cofactor molecules within a dense array is initially given priority. Nevertheless, constructions accommodating dense arrays of different cofactors, some well within 1 nm from each other, still presents a significant challenge for protein design.
... Coupling energy transfer to charge separation Cation and anion radicals are very efficient quenchers of excitation energy (Law and Cogdell 1998). The proximity of LH and RC pigments required for efficient EET increases the risk of forming such radicals particularly since the RC ET chains are designed to effectively carry away counter charges, thereby generating long-lived radicals. ...
The vast structural and functional information database of photosynthetic enzymes includes, in addition to detailed kinetic records from decades of research on physical processes and chemical reaction-pathways, a variety of high and medium resolution crystal structures of key photosynthetic enzymes. Here, it is examined from an engineer's point of view with the long-term goal of reproducing the key features of natural photosystems in novel biological and non-biological solar-energy conversion systems. This survey reveals that the basic physics of the transfer processes, namely, the time constraints imposed by the rates of incoming photon flux and the various decay processes allow for a large degree of tolerance in the engineering parameters. Furthermore, the requirements to guarantee energy and electron transfer rates that yield high efficiency in natural photosystems are largely met by control of distance between chromophores and redox cofactors. This underlines a critical challenge for projected de novo designed constructions, that is, the control of spatial organization of cofactor molecules within dense array of different cofactors, some well within 1 nm from each other.
Bacterial photosynthesis provides a simplified model system ideally for studying the basic mechanism of light-energy harvest and conversion. The early events in this process are carried out by two distinct components, the light-harvesting (LH)
complexes and the reaction center (RC)
. The LH complexes in purple photosynthetic bacteria are classified into two major types, the core LH1 complex that surrounds the RC and the peripheral LH2 complex that exists around the LH1. In addition to light-harvesting, the LH1 also plays a role in quinone (Q)
transport between the RC and quinone pool in the cell membrane. While several high-resolution structures are known for the RC and LH2, the structures of LH1 remained at low resolutions. Here, the crystal structure of a LH1-RC complex from thermophilic purple sulfur bacterium Thermochromatium tepidum is described. This complex is characterized by an enhanced thermostability and an absorption maximum at 915 nm for the LH1. These properties have been shown to be regulated by Ca2+ ions. The structure reveals a closed arrangement of LH1 complex around the RC, and the LH1 BChl a molecules form a partially overlapping ring with a shorter Mg–Mg spacing compared with that of B850 in LH2. Structural evidence is for the first time provided for the possible ubiquinone pathway in the closed LH1 complex. The Ca2+-binding sites are identified. Molecular mechanisms of quinone transport, Ca2+-regulation and interaction between LH1 and RC are discussed.
Antenna proteins from purple photosynthetic bacteria are by far the best-understood photosynthetic light-harvesting proteins, and among the best-characterized membrane proteins in any biological field. The photosynthetic membrane of purple bacteria is an exceptional case of a membrane for which structural information is available on the partner proteins involved in a biological process. The bacterial light-harvesting system constitutes an ideal source of experimental results for confronting theories to explain the functioning of these biological molecules in elementary physical and chemical terms, and for exploring light capture and transfer mechanisms at the level of the whole membrane. This chapter reviews the different aspects of our current knowledge on light-harvesting proteins from purple bacteria. A special emphasis is given to the biochemical properties of these complexes, their natural diversity, and the details of the known structures. The most recent results on the physical mechanisms that underlie their electronic properties, and on the cascade of the ultrafast excitation transfers that follow the absorption of the solar light are summarized. These are discussed in the light of the different models and calculations that have been performed from the crystal structure.
The light-harvesting protein complex 1 (LH1) of the purple bacterium Rhodobacter sphaeroides exhibits EPR signals upon treatment with oxidizing reagents such as potassium femicyanide. These signals are assigned to radical cations of the LH1's bacteriochlorophyll pigments, B880.(+). An intriguing feature of the B8800.(+) EPR spectrum is the narrow line width exhibited relative to in vitro monomeric BChla.(+) and the primary donor radical cation of the photosynthetic reaction center's "special pair", P.(+). In this paper, we investigate the temperature and oxidant concentration dependence of the B880.(+) EPR spectrum with the aim of elucidating the mechanism for spectral line narrowing. The experimental data are interpreted in terms of EPR line narrowing that accompanies charge migration and spin exchange. For charge migration, the line-narrowing models are driven by standard, nonadiabatic electron transfer assisted by vibronic coupling. The results are consistent with a hypothesis that, in LH1, the EPR spectral shape is dominated by electron transfer instead of spin exchange. In addition, the electronic and energetic factors governing the inter-BChla cryogenic charge transport are explored. Using standard treatments, large reorganization energy and weak electronic coupling are obtained for the charge migration process. The EPR results support the view that highly delocalized radical cation states similar to that observed for the primary donor BChlas of the special pair of the photosynthetic reaction center do not occur in oxidized LH1 complexes in the 6-300 K temperature range. However, the EPR results are compatible with a highly asymmetrical version of the special pair. The unrealistically high value of reorganization energy for electron transfer is attributed to treating the charge migration process as if electron transfer were homogeneous. A more realistic value of reorganization energy is predicted to result if free-energy heterogeneity were to be included in modeling electron transfer in LH1.
Exciton dynamics in the B850 and B875 bands of isolated complexes of Rhodopseudomonas acidophila (strain 10 050 and 7050) and in the B875 band of isolated complexes of Rhodobium marinum were investigated by means of accumulated photon echo and pump−probe techniques at different temperatures and wavelengths. For all three systems, the optical dephasing time T2 was found to be very similar: at 4.2 K, T2 is 116 and 106 ps for the B850 and B875 bands of Rhodopseudomonas acidophila, respectively, and 93 ps for the B875 band of Rhodobium marinum. The rapid dephasing, which displays glassy character, is a consequence of the strong pigment−protein interactions that arise through the rather short distances in these complexes. The observed dephasing time at the red edge of the B850 band of Rhodopseudomonas acidophila at 4.2 K reveals the existence of spectral diffusion in this system. From the wavelength dependence of the pump−probe signal in the B875 LH1 band of Rhodopseudomonas acidophila at 3 K it is concluded that energy transfer between energetically inequivalent LH1 rings occurs on a time scale of several tens picoseconds, while energy trapping takes place in about 250 ps.
We studied the time-resolved fluorescence of isolated RC-LH1 complexes from Rhodopseudomonas palustris as a function of the photon fluence and the repetition rate of the excitation laser. Both parameters were varied systematically over three orders of magnitude. Based on a microstate description we developed a quantitative model for RC-LH1 and obtained very good agreement between experiments and elaborate simulations based on a global master equation approach. The model allows us predict the relative population of RC-LH1 complexes with the special pair in the neutral state or in the oxidized state P(+), and those complexes that lack a reaction centre.
Single-molecule spectroscopy, by getting rid of unwanted ensemble averaging effects, has proved to be a very valuable tool in the research of individual photosynthetic light-harvesting (LH) complexes. Yet to learn about real photosynthetic processes the minimal unit to study is a single photosynthetic membrane complete with all elements of its machinery. In the present work the ambient-temperature fluorescence spectra of excitons in lone intracytoplasmic (IC) photosynthetic membrane vesicles of the wild type purple bacterium Rhodobacter sphaeroides that involve peripheral (LH2) and core (RC-LH1-PufX) antenna pigment-protein complexes were investigated under continuous-wave laser excitation into the Qx absorption band of the bacteriochlorophyll-a (BChl) chromophores at 594 nm. In parallel, the spectra of mutant membrane vesicles occupied by just one type of complexes (either LH2 or RC-LH1-PufX), and the spectra of individual purified LH2 and RC-LH1-PufX complexes were measured. The fluorescence from full IC membranes shows high sensitivity to excitation intensity, being varied between 0.1 and 2 kW/cm2. At low to moderate excitation intensities the spectra of IC membranes could be well reproduced by its component spectra, the ratio of the spectra related to peripheral and core complexes being the only adjustable parameter. The spectra of both intact chromatophores and individual membrane components recorded over 1-50 s experimental time frames are robust, strongly suggesting that large spectral fluctuations hardly play a role in the functional photosynthetic process. The significant, up to 14 times, variation of the LH2 and LH1 emission ratio observed in individual IC membranes could be related to variations in the stoichiometric ratio of the peripheral and core complexes. Evidence was found for the presence of LH2 parts that are detached from efficient energy transfer pathways. Upon strong and prolonged illumination the membrane spectra reveal significant permanent modifications. These alterations, which mostly concern peripheral antenna complexes, were shown to be due to photo-oxidation of various numbers of BChl molecules in the B850 compartment of LH2.
Effect of chemical oxidation by ferricyanide on bacteriochlorophyll a (BChl a) in the Fenna-Matthews-Olson protein (FMO) was studied using absorbance and fluorescence spectroscopy at ambient and cryogenic temperatures. Partially selective oxidation of pigments bound to the antenna complex was achieved and the probable absorption wavelength corresponding to the recently discovered bacteriochlorophyll No. 8 of 806 nm was obtained by comparative analysis of the effect of chemical oxidation and the effect of different isolation procedures. Formation of a stable product identified as a chlorophyll a derivative occurred upon chemical oxidation. This new pigment remained bound within the pigment-protein complex, and exhibited an efficient energy transfer to BChl a. Furthermore, complex effects of the pigment oxidation upon the fluorescence yield of the FMO protein were observed. Utility of this approach based on chemical modifications for the investigation of the native regulatory mechanisms involved in the energy transfer in the FMO protein is discussed.
We study the photo-oxidation of bacteriochlorophylls (BChls) in
peripheral light harvesting complexes (LH2) from rhodobacter
sphaeroides by using the steady absorption and the femtosecond
pump–probe measurement, to realize the detailed dynamics of LH2 in the
presence of photo-oxidation. The experimental results reveal that
BChl-B850 radical cations may act as an additional channel to compete
with the unoxidized BChl-B850 molecules for rapidly releasing the
excitation energy, while the B800→B850 energy transfer rate is almost
unaffected in the oxidation process.
In this research we used diverse nanofabrication techniques in order to direct the assembly on micro- and
nanostructured surfaces of purified units from the photosynthetic unit of purple bacteria. This allowed us to explore
the unique energy transfer properties of light harvesting complexes by producing biomolecular photonic wires. We
developed an approach based on the combination of site-directed mutagenesis, nanoimprint lithography and
multivalent host-guest interactions for the realization of engineered ordered functional arrays of purified components
of the photosynthetic system, the membrane-bound LH2 complex. In addition to micrometer-scale patterned
structures, we demonstrated the use of nanometer-scale hard NIL stamps to generate functional protein arrays
approaching molecular dimensions. We also report the first observation of long-range transport of excitation energy
within a bio-mimetic molecular light-guide constructed from LH2 antenna complexes organized vectorially into
functional nanoarrays. Fluorescence microscopy of the emission of light after local excitation with a diffractionlimited
light beam reveals long-range transport of excitation energy over micrometer distances, which is much larger
than required in the parent bacterial system.
Other biological systems used were visible fluorescent proteins and α-synuclein, an intrinsically unfolded protein
associated with Parkinson’s disease. We report for the first time the directed assembly and characterization of FRET
pairs on micrometer dimension patterned surfaces. In order to characterize the biological assemblies on the surfaces
AFM imaging in combination with optical imaging (spectral fluorescence microscopy and lifetime measurements)
were performed in liquid conditions.
Forty femtosecond pump and probe investigations in the 870 nm absorption band of the reconstituted core antenna, LH1, from Rhodobacter sphaeroides, in which varying amounts of Ni– bacteriopheophytin replace part of the native bacteriochlorophyll, show a tremendous shortening of the ground state recovery time with increasing amount of exchanged pigments. In the Ni–bacteriopheophytin containing antenna, a 60 fs deactivation channel has been found, which originates from a one-exciton state delocalized over the whole LH1 of about 20 pigment molecules. The 60 fs channel is interpreted as internal conversion in Ni–bacteriopheophytin.
An additional component in the purified core light-harvesting complex (LH1) from wild-type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of -polypeptide by MALDI-TOF mass spectrometry. This component appears as a slightly earlier-eluting peak in the RP-HPLC chromatogram compared with the authentic -polypeptide. The oxidation site has been determined to be the N-terminal methionine residue by high-resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form. Interconversion between the oxidized and authentic -polypeptides has been confirmed by selective oxidation and reduction. The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with β-polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit. Both the ability and the stability for the samples using the oxidized -polypeptide are moderately reduced, indicating that the oxidation-induced conformational change in the N-terminal domain of -polypeptide may affect the pigment-binding environment through a long-range interaction. The MALDI-TOF mass results also reveal that the N-terminus of -polypeptide is formylated and no phosphorylation has occurred in this polypeptide.
In this paper, we introduce the photo-induced ultrafast dynamics taking place in the peripheral light harvesting antenna LH2
from purple bacteria Rhodobacter sphaeroides by using absorption, fluorescence emission and ultrafast spectroscopic techniques. Three kinds of LH2 samples, pH treated
LH2 (complete removal of B800 pigments), carotenoid mutated LH2 (GM 309) and electrochemical oxidation treated LH2 were used
in comparison with native LH2 to investigate the mechanism of photo-induced ultrafast energy transfer within the LH2 complex.
The function of photosynthetic light harvesting complexes (LHCs) comprises absorption and regulated excitation energy transfer
(EET) to the photochemical reaction centers (RCs). Photosynthesizing organisms have developed a variety of LHCs but, apart
from phycobilins in cyanobacteria and certain algae, use only two types of pigments, (bacterio)chlorophylls ((B)Chl) and carotenoids.
Here we report the first observation of long-range transport of excitation energy within a biomimetic molecular nanoarray constructed from LH2 antenna complexes from Rhodobacter sphaeroides. Fluorescence microscopy of the emission of light after local excitation with a diffraction-limited light beam reveals long-range transport of excitation energy over micrometer distances, which is much larger than required in the parent bacterial system. The transport was established from the influence of active energy-guiding layers on the observed fluorescence emission. We speculate that such an extent of energy migration occurs as a result of efficient coupling between many hundreds of LH2 molecules. These results demonstrate the potential for long-range energy propagation in hybrid systems composed of natural light harvesting antenna molecules from photosynthetic organisms.
Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties. The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-beta-glucopyranoside (OG). Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll. By adding increasing amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap. In contrast to the nearly unchanged absorption, the presence of [Ni]-BChl in LH1 markedly affects the emission properties. Incorporation of only 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, respectively. The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 +/- 1 BChl molecules.
Five carotenoids existing in the purple bacterium of Rhodobium marinum, lycopene, anhydrorhodovibrin, spirilloxanthin, rhodopin, and rhodovibrin, were isolated and purified. Their configurations in the chromophore region and conformations of the terminal part were determined by 1D, 2D 1H and 13C NMR spectroscopy. The semiempirical quantum chemical calculation AM1 was subsequently performed using the rough 3-D structures established by NOE correlations as an initial input. The final optimized structures are coincident with 1H-1H NOE correlations and match with the X-ray crystallographic data of carotenoids. The calculation results show that chemically symmetrical carotenoids have a Ci point group. The Ci point group of molecules was destroyed by asymmetrical terminal part although the polyene chain still keeps it roughly. The polyene region of investigated carotenoids are in all-trans with slightly twisted in-plane and slight out-plane forming s-shape carbon backbone due to the spatial interaction of the methyl groups. Terminal parts, on the other hand, have several stable conformers due to the freely rotatable single bonds, but they prefer to take extended conformations.
The design of photosynthetic systems reflects the length scales of the fundamental physical processes. Energy transfer is rapid at the few angstrom scale and continues to be rapid even at the 50-Å scale of the membrane thickness. Electron tunneling is nearly as rapid at the shortest distances, but becomes physiologically too slow well before 20 Å. Diffusion, which starts out at a relatively slow nanosecond time scale, has the most modest slowing with distance and is physiologically competent at all biologically relevant distances. Proton transfer always operates on the shortest angstrom scale. The structural consequences of these distance dependencies are that energy transfer networks can extend over large, multisubunit and multicomplex distances and take leaps of 20 Å before entering the domain of charge separating centers. Electron transfer systems are effectively limited to individual distances of 15 Å or less span the 50 Å dimensions of the bioenergetic membrane by use of redox chains. Diffusion processes are generally used to cover the intercomplex electron transfer distances of 50 Å and greater and tend to compensate for the lack of directionality by restricting the diffusional space to the membrane or the membrane surface, and by multiplying the diffusing species through the use of pools. Proton transfer reactions act over distances larger than a few angstroms through the use of clusters or relays, which sometimes rely on water molecules and which may only be dynamically assembled.
The oxidation of bacteriochlorophylls (BChls) in peripheral light-harvesting complexes (LH2) from Rhodobacter sphaeroides was investigated by spectroelectrochemistry of absorption, fluorescence emission, and femtosecond (fs) pump-probe, with the aim obtaining information about the effect of in situ electrochemical oxidation on the pigment-protein arrangement and energy transfer within LH2. The experimental results revealed that: (a) the generation of the BChl radical cation in both B800 and B850 rings dramatically induced bleaching of the characteristic absorption in the NIR region and quenching of the fluorescence emission from the B850 ring for the electrochemical oxidized LH2; (b) the BChl-B850 radical cation might act as an additional channel to compete with the unoxidized BChl-B850 molecules for rapidly releasing the excitation energy, however the B800-B850 energy transfer rate remained almost unchanged during the oxidation process.
The ability of chlorophylls to coordinate ligands is of fundamental structural importance for photosynthetic pigment-protein complexes, where in virtually all cases the pigment is thought to be in a pentacoordinated state. In this study, the correlation of the Q(X) transition energy with the coordination state of the central metal in bacteriochlorophyll is applied in investigating the pigment coordination state in bacterial photosynthetic antenna LH1. To facilitate a detailed spectral analysis in the Q(X) region, carotenoid-depleted forms of LH1 are prepared and model LH1 are constructed with non-native carotenoids having blue-shifted absorption. The deconvolution of the Q(X) envelope in LH1 reveals that the band is the sum of two transitions, which peak near 590 and 607 nm, showing that a significant fraction (up to 25%) of hexacoordinated bacteriochlorophyll is present in the complex. The hexacoordination can be seen also in LH1 antennae from other species of purple photosynthetic bacteria. It seems correlated with the LH1 aggregation state and probably is a consequence of the structural flexibility of the assembled complex. The sixth ligand probably originates from the apoprotein and seems not to affect the chromophore core size. These findings show that in light-harvesting complexes a hexacoordinated state of bacteriochlorophyll is not uncommon. Its presence may be relevant to a correct assembly of the antenna and have functional consequences, as it results in a splitting of the pigment S2 excited state (Q(X)), i.e., the carotenoid excitation acceptor state, what might affect intracomplex carotenoid-to-bacteriochlorophyll energy transfer.
This review describes the structures of the two major integral membrane pigment complexes, the RC-LH1 'core' and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to 'power' the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.
Single molecule spectroscopy was applied to study the optical properties of native and refolded peridinin-chlorophyll-protein (PCP) complexes. The native system is a trimer with six chlorophyll a (Chl a) molecules, while the refolded one contains two Chl a and resembles structurally and spectroscopically the PCP monomer. The fluorescence emission of single PCP complexes strongly broadens with increasing excitation power. Simultaneously, the distribution of fluorescence maximum frequencies is also broadened. These spectral changes are attributed to photoinduced conformational changes of the protein that influence the fluorescence of embedded chromophores. Comparison of fluorescence intensities measured for PCP complexes in two different solvents indicates that the native PCP trimers are preserved in EDTA Tris buffer, while in PVA polymer matrix only monomers are stable.
The crystal structure of the light-harvesting antenna complex (LH2) from Rhodopseudomonas acidophila strain 10050 shows that the active assembly consists of two concentric cylinders of helical protein subunits which enclose the pigment molecules. Eighteen bacteriochlorophyll a molecules sandwiched between the helices form a continuous overlapping ring, and a further nine are positioned between the outer helices with the bacteriochlorin rings perpendicular to the transmembrane helix axis. There is an elegant intertwining of the bacteriochlorophyll phytol chains with carotenoid, which spans the complex.
Two-dimensional crystals from light-harvesting complex I (LHC I) of the purple non-sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent-solubilized protein complexes. Frozen-hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha- and the beta-polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.
Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the
complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties
were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced
with a lifetime of 1 ± 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the
18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy
transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all
molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed
to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter
lifetimes caused by excitation energy traps that are only ≈3% efficient. On repeated excitation some molecules entered a noisy
state where the fluorescence switched on and off with a correlation time of ≈0.1 s. About 490 molecules were examined.
The thylakoid membrane of Rhodopseudomonas viridis contains extensive, regular arrays of photoreceptor complexes arranged on a hexagonal lattice with a repeat distance of 130 A. Single membrane sheets were obtained by mild treatment of the thylakoid fraction with the detergent Triton X-100. Heavy metal shadowing and electron microscopy of isolated thylakoids indicated a strong asymmetry of the membrane, showing a smooth plasmic and a rough exoplasmic side. Fourier processing of rotary-shadowed specimens showed the different surface relief on both sides of the membrane. Structural units on both sides were roughly circular and showed 6-fold symmetry at a resolution close to 20 A. The structural unit was characterised by a central core that seemed to extend through the membrane, protruding on the exoplasmic side. The core was surrounded by a ring showing 12 subunits on the plasmic side. Rotary-shadowed as well as negatively-stained membranes indicated a handedness of the structure. Treatment of thylakoid vesicles with higher detergent concentrations yielded a fraction of particles showing the same features as Fourier maps of the structural units. The isolated particles therefore appeared to represent structurally intact units of photosynthesis.
Intense continuous illumination of purified chromatophores from carotenoidless mutant Rhodopseudomonas sphaeroides results in progressive photooxidative loss of the near infrared absorption band near 860 nm assigned to antenna bacteriochlorophyll. The quantum yield of this reaction is low, approximately 1.7 x 10(-5). The loss in near infrared absorption is accompanied by a proportional shift in the absorption maximum to shorter wavelengths. The double circular dichroism feature in the near infrared decreases at a faster rate than does the absorbance. These results are explained by a model in which the antenna bacteriochlorophyll, initially associated as dimers (lambda(max) = 860.2 nm), is progressively converted to the monomeric state (lambda(max) = 851.9 nm). The wavelength shift is attributed to disruption of exciton coupling in the dimer. Acetone/methanol extraction indicates that the maximum molar extinction coefficients of the dimer and monomer do not differ by more than 4%. The occurrence of an absorption maximum at 852 nm for monomeric bacteriochlorophyll in a protein complex demonstrates that it is not necessary to invoke aggregation of the chromophores as the origin of the shift from 770 nm in typical organic solvents.
Two‐dimensional crystals from light‐harvesting complex I (LHC I) of the purple non‐sulfur bacterium Rhodospirillum rubrum have been reconstituted from detergent‐solubilized protein complexes. Frozen‐hydrated samples have been analysed by electron microscopy. The crystals diffract beyond 8 A and a projection map was calculated to 8.5 A. The projection map shows 16 subunits in a 116 A diameter ring with a 68 A hole in the centre. These dimensions are sufficient to incorporate a reaction centre in vivo. Within each subunit, density for the alpha‐ and the beta‐polypeptide chains is clearly resolved, and the density for the bacteriochlorophylls can be assigned. The experimentally determined structure contradicts models of the LHC I presented so far.
11. Such near infrared changes are very similar to those observed upon light-induced or dark oxidation of photoreaction center bact- eriochlorophyll (the primary donor of bacterial photosynthesis), i.e., a bleaching at 865 nm and increase in optical density at 1245 nm [2]. Besides, the oxidized primary donor exhibits an ESR signal with a Gaussian lineshape, a g-value of 2.0025 and a peak-to-peak derivative linewidth of 9.5 G 131. In view of the similarities existing between the near infrared changes that follow oxidation of antenna and photoreaction center bacteriochlorophylls, it seemed interesting to investigate whether the oxi- dized antenna pigment is a paramagnetic species which can also be detected by ESR spectroscopy. As reported here, a nearly Gaussian ESR signal with a g-value of 2.0025 and a linewidth of 3.8 G appears to be due to the oxidized bacteriochlorophyll con- stituent which exhibits the 1230-nm band. Such a constituent seems to account only for - 1/3rd of total antenna bacteriochlorophyll and has an ap- parent midpoint redox potential of -555 mV (pH 8.0). A preliminary report of this work has been presented in [4].
Four new isolates from different saline habitats are described as the new species Rhodopseudomonas marina. Cells are short, motile rods with intracytoplasmic membranes lying parallel to the cytoplasmic membrane. Bacteriochlorophyll a, and carotenoids of the normal spirilloxanthin series, are the photosynthetic pigments. The new species grows photo-trophically in the light under anaerobic conditions, but is also capable of growing in the dark under microaerophilic or anaerobic conditions, depending on the carbon source. Under phototrophic conditions a wide variety of organic carbon sources are used. Excellent growth with the sugar alcohols mannitol and sorbitol is the most significant aspect. Only rather low concentrations of sulfide are tolerated, although sulfide tolerance is enhanced in the presence of small amounts of yeast extract. Sulfide is not oxidized to sulfate; thiosulfate and elemental sulfur are the only detectable oxidation products. The different strains require 1-5% NaCl for optimal growth. The DNA base ratio is 61.5-63.8 mol% G + C. Rhodopseudomonas marina can be differentiated from all known Rhodospirillaceae species by its lipid and quinone composition.
The molecular structure of the photosynthetic reaction centre from Rhodopseudomonas viridis has been elucidated using X-ray crystallographic analysis. The central part of the complex consists of two subunits, L and M, each of which forms five membrane-spanning helices. We present the first description of the high-resolution structure of an integral membrane protein.
Electron micrographs of photosynthetic membranes of the BChla-containing bacterium Rp. marina showed a quasi-crystalline structure. The photoreceptor units are arranged in a hexagonal lattice with a reaction center to reaction center distance of 102 +/- 3 A. Purified B880-complex was concentrated up to an OD880 of 60 which induced the formation of large protein vesicles. The protein complexes within these vesicles were highly ordered and showed a hexagonal lattice with the same center to center distance of 102 +/- 3 A as was observed in the native membranes. Image processing of the micrographs revealed a ring-like structure of the B880-complex at 26 A resolution and suggests that the B880-complex consists of 5 or 6 subunits. For the first time it can be shown that an isolated core-complex is in a stable, ring-like structure even without the reaction center which is supposed to be located in the middle of the B880-ring. The data indicate that the isolated B880-complex exhibits the same structure as in the native membrane.
The BChla-containing B880-complex (core-complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880-complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium-sulfate precipitation with about 50% recovery. The B880-complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880-complex with the detergent n-octyl-beta-glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The alpha- to beta-polypeptide ratio and the alpha- or beta-polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820-complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880-complex of Rp. marina consists of 24 polypeptides and the B820-complex of four polypeptides. Six B820-complexes or possible subunits could form the B880-complex. On the basis of these data we propose a model for the structure of BChla containing core-complexes.
The three-dimensional structure of the cofactors of the reaction center of Rhodobacter sphaeroides R-26 has been determined by x-ray diffraction and refined at a resolution of 2.8 A with an R value of 26%. The main features of the structure are similar to the ones determined for Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 5, 2445-2451]. The cofactors are arranged along two branches, which are approximately related to each other by a 2-fold symmetry axis. The structure is well suited to produce light-induced charge separation across the membrane. Most of the structural features predicted from physical and biochemical measurements are confirmed by the x-ray structure.
Certain redox properties of bacteriochlorophyll alpha were used to probe the structure of several light-harvesting pigment-protein complexes or holochromes. To attribute redox properties unequivocally to a given holochrome, we worked with purified holochromes. We developed purification procedures for the B880 holochromes from Rhodospirillum rubrum, Rhodopseudomonas sphaeroides and Ectothiorhodospira sp. and for the B800-850 holochromes from the latter two species. In all these holochromes, bacteriochlorophyll alpha could be oxidized by ferricyanide as witnessed by the bleaching of their near-infrared absorption bands. However, only in B880 holochromes was this oxidation reversible. Another important difference between the B800-850 and the B880 holochromes is that oxidation of the latter gives rise to a g = 2.0025 electron paramagnetic resonance (EPR) signal with linewidth varying, according to species, from 0.37 mT to 0.48 mT. Both the reversible EPR signal and absorption changes titrate with a midpoint redox potential (pH 8.0) of approximately 570 mV. Linewidth narrowing can be interpreted by delocalization of the free electron spin over approximately 12 bacteriochlorophyll molecules. While the B880 holochromes from the three species considered had indistinguishable redox properties, the B800-850 holochromes differed from one another by their circular dichroic spectra and by the relative ease of oxidation of their 800-nm and 850-nm bands. This indicates that, contrary to the B880 holochromes, the B800-850 holochromes may not form a homogeneous class.
Background:
The light-harvesting complexes II (LH-2s) are integral membrane proteins that form ring-like structures, oligomers of alpha beta-heterodimers, in the photosynthetic membranes of purple bacteria. They contain a large number of chromophores organized optimally for light absorption and rapid light energy migration. Recently, the structure of the nonameric LH-2 of Rhodopseudomonas acidophila has been determined; we report here the crystal structure of the octameric LH-2 from Rhodospirillum molischianum. The unveiling of similarities and differences in the architecture of these proteins may provide valuable insight into the efficient energy transfer mechanisms of bacterial photosynthesis.
Results:
The crystal structure of LH-2 from Rs. molischianum has been determined by molecular replacement at 2.4 A resolution using X-ray diffraction. The crystal structure displays two concentric cylinders of sixteen membrane-spanning helical subunits, containing two rings of bacteriochlorophyll-a (BChl-a) molecules. One ring comprises sixteen B850 BChl-as perpendicular to the membrane plane and the other eight B800 BChl-as that are nearly parallel to the membrane plane; eight membrane-spanning lycopenes (the major carotenoid in this complex) stretch out between the B800 and B850 BChl-as. The B800 BChl-as exhibit a different ligation from that of Rps. acidophila (aspartate is the Mg ligand as opposed to formyl-methionine in Rps. acidophila).
Conclusions:
The light-harvesting complexes from different bacteria assume various ring sizes. In LH-2 of Rs. molischianum, the Qy transition dipole moments of neighbouring B850 and B800 BChl-as are nearly parallel to each other, that is, they are optimally aligned for Föster exciton transfer. Dexter energy transfer between these chlorophylls is also possible through interactions mediated by lycopenes and B850 BChl-a phytyl tails; the B800 BChl-a and one of the two B850 BChl-as associated with each heterodimeric unit are in van der Waals distance to a lycopene, such that singlet and triplet energy transfer between lycopene and the BChl-as can occur by the Dexter mechanism. The ring structure of the B850 BChl-as is optimal for light energy transfer in that it samples all spatial absorption and emission characteristics and places all oscillator strength into energetically low lying, thermally accessible exciton states.
Oligomers of [E,E] BChl CF (8, 12-diethyl bacteriochlorophyll c esterified with farnesol (F)) and [Pr,E] BChl CF (analogously, M methyl, Pr propyl) in hexane and aqueous detergent or lipid micelles were studied by means of steady-state absorption, time-resolved fluorescence, and electron spin resonance spectroscopy. The maximum absorption wavelength, excited-state dynamics, and electron spin resonance (EPR) linewidths are similar to those of native and reconstituted chlorosomes of Chlorobium tepidum. The maximum absorption wavelength of oligomers of [E,E] BChl CF was consistently blue-shifted as compared to that of [Pr,E] BChl CF oligomers, which is ascribed to the formation of smaller oligomers with [E,E] BChl CF than [Pr,E] BChl CF. Time-resolved fluorescence measurements show an excited-state lifetime of 10 ps or less in nonreduced samples of native and reconstituted chlorosomes of Chlorobium tepidum. Under reduced conditions the excited-state lifetime increased to tens of picoseconds, and energy transfer to BChl a or long-wavelength absorbing BChl c was observed. Oligomers of [E,E] BChl CF and [Pr,E] BChl CF in aqueous detergent or lipid micelles show a similar short excited-state lifetime under nonreduced conditions and an increase up to several tens of picoseconds upon reduction. These results indicate rapid quenching of excitation energy in nonreduced samples of chlorosomes and aqueous BChl c oligomers. EPR spectroscopy shows that traces of oxidized BChl c radicals are present in nonreduced and absent in reduced samples of chlorosomes and BChl c oligomers. This suggests that the observed short excited-state lifetimes in nonreduced samples of chlorosomes and BChl c oligomers may be ascribed to excited-state quenching by BChl c radicals. The narrow EPR linewidth suggests that the BChl c are arranged in clusters of 16 and 6 molecules in chlorosomes of Chlorobium tepidum and Chloroflexus aurantiacus, respectively.
A stoichiometric unit of the light-harvesting complex I and the reaction centre (LHI-RC complex) has been isolated from a carotenoid-less mutant of the purple non-sulphur bacterium Rhodospirillum rubrum by mild solubilization of photosynthetic membranes with the phospholipid detergent diheptylphosphatidylcholine. Dialysis of the isolated LHI-RC complexes in the presence of added dioleoyl-sn-phosphatidylcholine produced ordered two-dimensional crystals. Digital image processing revealed that the LHI-RC are packed together in a square lattice (a = b = 16.3 nm). The dimensions of the LHI ring are essentially identical with those determined from two-dimensional (2D) crystals of purified carotenoid-containing light-harvesting I complexes after analysis by cryo-electron microscopic techniques or from negatively stained 2D crystals of purified LHI complexes from a carotenoid-less mutant. Each LHI ring of the LHI-RC complex contains a central diffuse stain-excluding region, which is assigned to the reaction centre. The analysis of the LHI-RC 2D crystals strongly suggests that the geometry and subunit stoichiometry of the LHI ring is unaffected by the presence of a reaction centre that can probably assume various orientations within the ring.
The photosynthetic reaction center-light harvesting 1 complex from Rhodopseudomonas viridis was purified and reconstituted into two-dimensional crystals. The single-layered crystalline sheets with lattice parameters a=b=133.3 A and gamma=120 degrees were investigated by electron cryo-microscopy and the projection map at 10 A resolution was calculated. The opening diameter of the light-harvesting ring of 72 A is sufficient to allow slight movement of the reaction center within the ring. Based on characteristic features observed in the projection map, the mechanism of energy transfer from the light-harvesting 1 complex to the reaction center was discussed.
- I Gomez
- R Picorel
- J M Ramirez
- R Perez
- F F Del Campo
Gomez, I., Picorel, R., Ramirez, J.M., Perez, R. and del Campo,
F.F. (1982) Photochem. Photobiol. 35, 399^403.
- R K Clayton
Clayton, R.K. (1962) Photochem. Photobiol. 1, 201^210.
- I Gomez
- C Sieiro
- J M Ramirez
- S Gomez-Amores
- F F Del Campo
Gomez, I., Sieiro, C., Ramirez, J.M., Gomez-Amores, S. and del
Campo, F.F. (1982) FEBS Lett. 144, 117^120.
- R Picorel
- S Lefebvre
- G Gingras
Picorel, R., Lefebvre, S. and Gingras, G. (1984) Eur. J. Biochem.
142, 305^311.
- W Stark
- W Ku Ë Hlbrandt
- I Wildhaber
- E Wehrli
- K Mu Ë Hlethaler
Stark, W., Ku ë hlbrandt, W., Wildhaber, I., Wehrli, E. and
Mu ë hlethaler, K. (1984) EMBO J. 3, 777^783.
- R U Meckenstock
- K Krusche
- R A Brunisholz
- H Zuber
- I Yamasaki
- T Odahara
- K Mitsuoka
- Y Fujiyoshi
- K Murata
Meckenstock, R.U., Krusche, K., Brunisholz, R.A. and Zuber,
H. (1992) FEBS Lett. 311, 135^138.
[10] Ikedu-Yamasaki, I., Odahara, T., Mitsuoka, K., Fujiyoshi, Y.
and Murata, K. (1998) FEBS Lett. 425, 505^508.
- R Van Grondelle
van Grondelle, R. (1985) Biochim. Biophys. Acta 811, 147^195.
- R U Meckenstock
- R A Brunisholz
- H Zuber
Meckenstock, R.U., Brunisholz, R.A. and Zuber, H. (1992)
FEBS Lett. 311, 128^134.
- J Koepke
- X C Hu
- C Muenke
- K Schulten
- H Michel
Koepke, J., Hu, X.C., Muenke, C., Schulten, K. and Michel, H.
(1996) Structure 4, 581^597.
- I Ikedu-Yamasaki
- T Odahara
- K Mitsuoka
- Y Fujiyoshi
- K Murata
Ikedu-Yamasaki, I., Odahara, T., Mitsuoka, K., Fujiyoshi, Y.
and Murata, K. (1998) FEBS Lett. 425, 505^508.