Dorte B. Steensgaard

Fast energy transfer between BChl d and BChl c in chlorosomes of the green sulfur bacterium Chlorobium limicola.

We have studied energy transfer in chlorosomes of Chlorobium limicola UdG6040 containing a mixture of about 50% bacteriochlorophyll (BChl) c and BChl d each. BChl d-depleted chlorosomes were obtained by acid treatment. The energy transfer between the different pigment pools was studied using both steady-state and time-resolved fluorescence spectroscopy at room temperature and low temperature. The steady-state emission of the intact chlorosome originated mainly from BChl c, as judged by comparison of fluorescence emission spectra of intact and BChl d-depleted chlorosomes. This indicated that efficient energy transfer from BChl d to BChl c takes place. At room temperature BChl c/d to BChl a excitation energy transfer (EET) was characterized by two components of 27 and 74 ps. At low temperature we could also observe EET from BChl d to BChl c with a time constant of approximately 4 ps. Kinetic modeling of the low temperature data indicated heterogeneous fluorescence kinetics and suggested the presence of an additional BChl c pool, E790, which is more or less decoupled from the baseplate BChl a. This E790 pool is either a low-lying exciton state of BChl c which acts as a trap at low temperature or alternatively represents the red edge of a broad inhomogeneous absorption band of BChl c. We present a refined model for the organization of the spatially separated pigment pools in chlorosomes of Cb. limicola UdG6040 in which BChl d is situated distal and BChl c proximal with respect to the baseplate.

Effect of alkaline treatment on bacteriochlorophyll a , quinones, and energy transfer in chlorosomes from Chlorobium tepidum and Chlorobium phaeobacteroides

Abstract— Chlorosomes isolated from two types of green sulfur bacteria, Chlorobium tepidum which contains bacteriochlorophyll c (BChl c) and the BChl e‐containing Chlorobium phaeobacteroides, were subjected to alkaline treatment (pH 12.7 at 40°C for 20 min). This caused selective degradation of BChl a, whereas BChl c or e were not affected. Chlorobiumquinone in the Chlorosomes was partially degraded by the alkaline treatment but menaquinone was unchanged. Fluorescence decay kinetics showed that alkaline treatment disrupted energy transfer from BChl c or e to BChl a under reducing conditions. However, this did not give rise to any substantial increase in the excited state lifetime of BChl e in C. phaeobacteroides Chlorosomes, while for C. tepidum a decrease in the BChl c lifetime was found. The steady‐state fluorescence of chlorosomes is highly dependent on the redox potential such that emission is quenched in oxidizing environments. Alkaline treatment diminished this quenching effect and caused a doubling in the BChl c or e emission intensity under aerobic conditions. Single‐photon timing experiments confirmed that alkaline treatment inhibits the energy trapping process operative under aerobic conditions. These effects of alkaline treatment on the fluorescence intensity and decay kinetics are likely to be related to the depletion in BChl a or in Chlorobiumquinone or a combination of these.

Evidence for spatially separate bacteriochlorophyll c and bacteriochlorophyll d pools within the chlorosomal aggregate of the green sulfur bacterium Chlorobium limicola

We have studied the organization of the bacteriochlorophylls (BChl) in isolated chlorosomes of the green sulfur bacterium Chlorobium limicola UdG6040 containing about 50% BChl d and BChl c each. When the chlorosomes are treated in acidic buffer (pH 3.0) two phases in the conversion from BChl to bacteriopheophytin (BPhe) are observed as evidenced by the changes in the absorption spectrum. In the early phase the pheophytinization of BChl d occurs much faster than that of BChl c. In the later phase BChl c and BChl d are converted at similar rates. The delayed BChl c conversion observed in intact chlorosomes is interpreted in terms of spatial separation within the same chlorosome that makes BChl d more accessible to reaction with acid than BChl c. This was supported by acid treatment of in vitro pigment-lipid aggregates which showed that the pheophytinization of aggregates consisting of only BChl c or BChl d takes place with the same rate. Moreover in mixed in vitro aggrega tes where BChl d and BChl c are supposed to be scrambled the two pigments are converted to BPhe simultaneously. Acid treatment of hexanol exposed chlorosomes indicates that the spatial separation of BChl d and BChl c within the chlorosomes is maintained even if the excitonic interaction between BChls has been disturbed by hexanol. Based on these findings it is suggested that BChl d and BChl c in the chlorosome are located distal and proximal, respectively, relative to the chlorosome baseplate.

Evidence from Solid State NMR Correlation Spectroscopy for two Interstack Arrangements in the Chlorosome Antenna System

Solid state cross-polarization (CP) MAS NMR dipolar correlation spectroscopy is a rapidly growing technique that can provide structural information of systems that are inaccessible for X-ray diffraction techniques [1]. In particular, we have implemented correlation spectroscopy to provide a concept for structure determination and to study the chromophore arrangement in antenna systems. Homonuclear (13C-13C) dipolar correlation spectroscopy was used to study the stacking of bacteriochlorophyll c (Bchl c) in uniformly 13C enriched [U-13C] intact chlorosomes from Chlorobium tepidum [2]. It was possible to arrive at a full assignment of the solid state NMR carbon chemical shifts, from which a model for the stack and interstack of Bchl c in the chlorosomes could be resolved [1-3].

Magic Angle Spinning NMR of Photosynthetic Components

Selectively and multispin labelled cofactors, ligands, amino acids and proteins can be prepared and used for investigations with MAS NMR to determine structure and mechanisms of function of biological systems. In particular, solid state crosspolarization (CP) MAS NMR dipolar correlation spectroscopy is a rapidly growing technique that can provide structural information of systems that are inaccessible for X-ray diffraction techniques [1]. In our research programme, a principal application of the MAS NMR is in the studies of photosynthetic energy conversion systems. In terms of spin-off, the elucidation of the functional concepts behind photosynthetic charge separation in nature is expected to be of assistance in improving artificial photosynthetic devices like photovoltaic cells. Photosynthesis is the process that converts solar energy into chemical energy, thus providing the earth’s major renewable energy resources. Development of renewable energy supplies is an urgent need from economic, strategic and environmental perspectives. Current official guidelines and industrial programmes put a strong emphasis on biomass, which is from a fundamental scientific perspective a giant technological leap backwards since only a few percent of the solar energy is converted (Table I). Although the actual application of artificial molecular solar energy converters may still be years ahead, elucidation of physical and chemical concepts behind Nature’s efficient primary processes in photosynthetic energy conversion, such as optimal compartmentation of light-harvesting and photochemical energy conversion devices, balance between charge separation and neutral spin separation, will be of use to photovoltaics and molecular electronics research.

Structure and Function of Chlorosomes of Chlorobium Limicola UdG 6040 Containing Both Bchl c and Bchl d

The organization of light-harvesting pigments is essential for the funneling of excitation energy towards the photosynthetic reaction center. In green bacteria the light-harvesting chlorosomes are oblong flattened bodies attached the inner side of the cytoplasmic membrane. The chlorosome is surrounded by a lipid monolayer and filled with BChl c, d or e organized in large rod shaped aggregates. Moreover a small amount of BChl a is situated in the so-called base plate, the part of the lipid envelope facing the cytoplasmic membrane. The chlorosomal BChl a functions as an intermediate acceptor in the energy transfer from the bulk BChls towards the reaction centers [1].

Alkali Treatment of Green Bacterial Chlorosomes

Green sulfur bacteria and green filamentous bacteria use a structurally and functionally similar light-harvesting apparatus called the chlorosome (1). Within a lipid monolayer envelope the chlorosome contains thousands of bacteriochlorophylls (BChl) c, d or e. A minor amount of BChl a is also present and is probably associated with proteins which form a baseplate. This BChl a mediates excitation energy between the chlorosome and the photosynthetic reaction center.