C.N. Hunter

Temperature dependence of energy transfer from the long wavelength antenna BChl-896 to the reaction center in Rhodospirillum rubrum, Rhodobacter sphaeroides (w.t. and M21 mutant) from 77 to 177K, studied by picosecond absorption spectroscopy.

Decay of the bacteriochlorophyll excited state was measured in membranes of the purple bacteria Rhodospirillum (R.) rubrum, Rhodobacter (Rb.) sphaeroides wild type and Rb. sphaeroides mutant M21 using low intensity picosecond absorption spectroscopy. The excitation and probing pulses were chosen in the far red wing of the long wavelength absorption band, such that predominantly the minor antenna species B896 was excited. The decay of B896 was studied between 77 and 177K under conditions that the traps were active. In all species the B896 excited state decay is almost temperature independent between 100 and 177K, and probably between 100 and 300 K. In this temperature range the decay rates for the various species are very similar and close to 40 ps. Below 100 K this rate remains temperature independent in Rb. sphaeroides w. t. and M21, while in R. rubrum a steep decrease sets in. An analysis of this data with the theory of nuclear tunneling indicates an activation energy for the final transfer step from B896 to the special pair of 70cm-1 for R. rubrum and 30cm-1 or less for Rb. sphaeroides.

The photosynthesis gene cluster of Rhodobacter sphaeroides

The photosynthetic bacteria are at the forefront of the study of many aspects of photosynthesis, including photopigment biosynthesis, photosynthetic-membrane assembly, light-harvesting, and reaction center photochemistry. The facultative growth of some photosynthetic bacteria, their simple photosystems, and their ease of genetic manipulation have all contributed to advances in these areas. Amongst these bacteria, the purple non-sulfur bacterium Rhodobacter sphaeroides has emerged as, arguably, the leading contender for a model system in which to integrate the studies of all the different aspects of the assembly and function of the photosynthetic apparatus. Many of the genes encoding photosynthesis-related proteins are known to be clustered within a small region of the genome in this organism. As a further aid to studying the assembly and function of the photosystem of Rb. sphaeroides, the DNA sequence for a genomic segment containing this photosynthesis gene cluster (PGC) has been assembled from previous EMBL submissions and formerly unpublished data. The Rb. sphaeroides PGC is 40.7 kb in length and consists of 38 open reading frames encoding the reaction center H, L and M subunits, the α and β polypeptides of the light-harvesting I (B875) complex, and the enzymes of bacteriochlorophyll and carotenoid biosynthesis. PGCs are a feature of gene organization in several photosynthetic bacteria, and the similarities between the clusters of Rb. sphaeroides and Rb. capsulatus have been apparent for some time. Here we present the first comprehensive analysis of the PGC of Rb. sphaeroides, as well as a comparison with that of Rb. capsulatus.

Light harvesting, electron transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10 microA/cm(2) for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

Excited-state dynamics of mutated antenna complexes of purple bacteria studied by hole-burning

Absorption and fluorescence excitation spectra of various LH2 antenna complexes of two purple bacteria at low temperature (1.2 and 4.2 K) have been measured, and energy transfer rates within these complexes have been determined by spectra hole-burning. The systems studied were membranes of a wild-type strain of Rhodobacter sphaeroides , membrane samples from four LH2-only strains containing specifically mutated LH2 complexes of the same bacterium, and the isolated B800–820 complex of Rhodopseudomonas acidophila (strain 7050). The mutants exhibit blue-shifted B850 absorption bands with their spectral positions depending on the specific amino acid residues replaced in the α-polypeptide sequence. Energy transfer rates from B800 to B850 (or to their respectively blue-shifted bands) have been obtained by hole-burning experiments in the B800 band. The mutants of Rb. sphaeroides and the LH2 complex of Rps. acidophila yielded transfer times similar to those of the B800–850 complex of Rb. sphaeroides . These values, which for the various complexes vary between 1.7 and 2.5 ps in the wavelength region from 798 to 805 nm, do not decrease monotonically with the spectral distance between the bands. Various models based on Forsters energy transfer mechanism are discussed, of which only one is consistent with the results. In this model the energy is assumed to be transferred not directly from the Q y 0-0 band of B800 to that of the (blue-shifted) B850, but indirectly through the excitation of a vibrational mode.

Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. II. A low-temperature spectroscopy study of structural aspects of the pigment-protein conformation

Abstract Low-temperature absorbance, fluorescence, linear and circular dichroism spectra were measured for two site-specific mutants (βHis21 → and βArg29 → Glu) of the peripheral (B800–850) light-harvesting complex of Rb. sphaeroides in order to obtain information on the possible changes in the binding site of the bacteriochlorophyll a pigments. From the absorbance and fluorescence measurements we conclude that when βHis21, the putative ligand of the BChl 800 pigment, is replaced by serine the pigment is not incorporated in the complex. In addition, this modification induces a 4 nm red-shift of the B850 band. Linear and circular dichroism measurements indicate that the specific orientation of the BChl 850 pigments is retained despite the absence of a pigment in the B800 binding-site. A second mutant, in which the conserved arginine at position 29 on the same polypeptide is replaced by glutamate, was also studied. This replacement causes a significant blue shift as well as a marked broadening of of the B800 band, which indicates that this binding site is more heterogeneous in this mutant. The overall orientation however is not drastically changed and energy transfer from the B800 to B850 still takes place efficiently. We conclude that neither residue is exclusively involved in the binding pocket for BChl 800, but both βHis21 and βArg29 are important in determining the BChl 800 binding.

Probing the B800 bacteriochlorophyll binding site of the accessory light-harvesting complex from Rhodobacter sphaeroides using site-directed mutants. I: Mutagenesis, effects on binding, function and electrochromic behaviour of its carotenoids

Abstract The light-harvesting LH2 complex of Rhodobacter sphaeroides contains two amino acid residues, βHis21 and βArg29, which are conserved in all LH2 β-polypeptides of purple nonsulfur bacteria sequenced so far. These residues have been changed into serine and glutamic acid, respectively. Both mutations lead to severe changes in the spectroscopic characteristics of the antenna complex. Changing βArg29 into Glu results in a blue shift and a broadening of the B800 bacteriochlorophyll absorbance, suggesting a role of this residue in creating the binding pocket for B800 (see also Visschers et al. (1994) Biochim. Biophys Acta 1183, 483–490). Similar blue shifts, of approx. 6 nm, are also observed in the carotenoid absorbance peaks. This is accompanied by a large change in the electrochromic behaviour of the carotenoids, which suggests a major role of βArg29 in creating a local field near the responsive carotenoid. The second mutation, βHis21 to Ser, results in an inability to create a B800 domain. This mutation also causes changes in the carotenoid absorbance and electrochromic behaviour, suggesting a direct or indirect (via the bacteriochlorophyll B800 molecule) effect on the local dipole field of the sensitive carotenoid. Neither of the mutated complexes has lost the ability to bind carotenoids; in both complexes energy transfer from the carotenoids to B850 appears unaltered, indicating that all carotenoids can transfer energy directly to this bacteriochlorophyll, despite the loss of B800.

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe.

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c2 is approximately 480 pN with an interaction frequency of around 66xa0%. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.

Dimerization of core complexes as an efficient strategy for energy trapping in Rhodobacter sphaeroides.

In the purple phototrophic bacterium Rhodobacter sphaeroides, light harvesting LH2 complexes transfer absorbed solar energy to RC-LH1-PufX core complexes, which are mainly found in the dimeric state. Many other purple phototrophs have monomeric core complexes and the basis for requiring dimeric cores is not fully established, so we analysed strains of Rba. sphaeroides that contain either native dimeric core complexes or altered monomeric cores harbouring a deletion of the first 12 residues from the N-terminus of PufX, which retains the PufX polypeptide but removes the major determinant of core complex dimerization. Membranes were purified from strains with dimeric or monomeric cores, and with either high or low levels of the LH2 complex. Samples were interrogated with absorption, steady-state fluorescence, and picosecond time-resolved fluorescence kinetic spectroscopies to reveal their light-harvesting and energy trapping properties. We find that under saturating excitation light intensity the photosynthetic membranes containing LH2 and monomeric core complexes have fluorescence lifetimes nearly twice that of membranes with LH2 plus dimeric core complexes. This trend of increased lifetime is maintained with RCs in the open state as well, and for two different levels of LH2 content. Thus, energy trapping is more efficient when photosynthetic membranes of Rba. sphaeroides consist of RC-LH1-PufX dimers and LH2 complexes.

Lateral segregation of photosystem I in cyanobacterial thylakoids

Atomic force microscopy, optical microscopy, and structural modeling reveal extensive macromolecular arrays of cyanobacterial PSI complexes that allow some intertrimer energy migration. Photosystem I (PSI) is the dominant photosystem in cyanobacteria and it plays a pivotal role in cyanobacterial metabolism. Despite its biological importance, the native organization of PSI in cyanobacterial thylakoid membranes is poorly understood. Here, we use atomic force microscopy (AFM) to show that ordered, extensive macromolecular arrays of PSI complexes are present in thylakoids from Thermosynechococcus elongatus, Synechococcus sp PCC 7002, and Synechocystis sp PCC 6803. Hyperspectral confocal fluorescence microscopy and three-dimensional structured illumination microscopy of Synechocystis sp PCC 6803 cells visualize PSI domains within the context of the complete thylakoid system. Crystallographic and AFM data were used to build a structural model of a membrane landscape comprising 96 PSI trimers and 27,648 chlorophyll a molecules. Rather than facilitating intertrimer energy transfer, the close associations between PSI primarily maximize packing efficiency; short-range interactions with Complex I and cytochrome b6f are excluded from these regions of the membrane, so PSI turnover is sustained by long-distance diffusion of the electron donors at the membrane surface. Elsewhere, PSI-photosystem II contact zones provide sites for docking phycobilisomes and the formation of megacomplexes. PSI-enriched domains in cyanobacteria might foreshadow the partitioning of PSI into stromal lamellae in plants, similarly sustained by long-distance diffusion of electron carriers.

Light-Driven Enzymatic Reaction in Thermophilic Protochlorophyllide Oxidoreductase

Within the chlorophyll biosynthetic pathway, NADPH protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent trans addition of hydrogen across the C17–C18 double bond of the D -ring of protochlorophyllide (Pchlide) to produce chlorophyllide (Chlide). The fact that POR is light activated means the enzyme–substrate complex can be formed in the dark, removing the diffusive components out of the reaction. This chapter analyzes POR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 in the same way. Short time scan measurements allow the control of the amount of light an enzyme has seen, or how often it has cycled through the excited state, and monitoring the concomitant reaction kinetics. The main observations based on the experiment on thermophilic POR include ultrafast product formation in thermophilic POR catalysis, blue shift of Pchlide stimulated emission and its decay, and faster and more product Chlide formation occurs in samples that have been excited before.