Photochemical Reaction Center Research Articles

2P350 Identification of electron transfer proteins reducing photochemical reaction center of the filamentous anoxygenic phototrophic bacteria(43. Photosynthesis,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P350 Identification of electron transfer proteins reducing photochemical reaction center of the filamentous anoxygenic phototrophic bacteria(43. Photosynthesis,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

A New Type of Bacteriophytochrome Acts in Tandem with a Classical Bacteriophytochrome to Control the Antennae Synthesis in Rhodopseudomonas palustris

Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far-red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all (bacterio)phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 autophosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semiaerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.

Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant

The molecular architectures of photosynthetic complexes are rapidly becoming available through the power of X-ray crystallography. These complexes are comprised of antenna complexes, which absorb and transfer energy into photochemical reaction centers. Most reaction centers, found in both oxygenic and non-oxygenic species, are connected to transmembrane chlorophyll containing antennas, and the crystal structures of these antennas contain information on the structure of the entire complex as well as clear indications on their modes of functional association. In cyanobacteria and red alga, most of the Photosystem II associated light harvesting is performed by an enormous (3-7 MDa) membrane attached complex called the phycobilisome (PBS). While the crystal structures of many isolated components of different PBSs have been determined, the structure of the entire complex as well as its manner of association with Photosystem II can only be suggested. In this review, the structural information obtained on the isolated components will be described. The structural information obtained from the components provides the basis for the modeled reconstruction of this giant complex.

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 °C for 2 h with light at an intensity of 2,500 μmol photons m −2 s −1, the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.

Two-Step Mechanism of Photodamage to Photosystem II: Step 1 Occurs at the Oxygen-Evolving Complex and Step 2 Occurs at the Photochemical Reaction Center

Under strong light, photosystem II (PSII) of oxygenic photosynthetic organisms is inactivated, and this phenomenon is called photoinhibition. In a widely accepted model, photoinhibition is induced by excess light energy, which is absorbed by chlorophyll but not utilized in photosynthesis. Using monochromatic light from the Okazaki Large Spectrograph and thylakoid membranes from Thermosynechococcus elongatus, we observed that UV and blue light inactivated the oxygen-evolving complex much faster than the photochemical reaction center of PSII. These observations suggested that the light-induced damage was associated with a UV- and blue light-absorbing center in the oxygen-evolving complex of PSII. The action spectrum of the primary event in photodamage to PSII revealed the strong effects of UV and blue light and differed considerably from the absorption spectra of chlorophyll and thylakoid membranes. By contrast to the photoinduced inactivation of the oxygen-evolving complex in untreated thylakoid membranes, red light efficiently induced inactivation of the PSII reaction center in Tris-treated thylakoid membranes, and the action spectrum resembled the absorption spectrum of chlorophyll. Our observations suggest that photodamage to PSII occurs in two steps. Step 1 is the light-induced inactivation of the oxygen-evolving complex. Step 2, occurring after step 1 is complete, is the inactivation of the PSII reaction center by light absorbed by chlorophyll. We confirmed our model by illumination of untreated thylakoid membranes with blue and UV light, which inactivated the oxygen-evolving complex, and then with red light, which inactivated the photochemical reaction center.

Structural and Spectroscopic Properties of a Reaction Center Complex from the Chlorosome-Lacking Filamentous Anoxygenic Phototrophic BacteriumRoseiflexus castenholzii

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.

Optimization and evolution of light harvesting in photosynthesis: the role of antenna chlorophyll conserved between photosystem II and photosystem I.

The efficiency of oxygenic photosynthesis depends on the presence of core antenna chlorophyll closely associated with the photochemical reaction centers of both photosystem II (PSII) and photosystem I (PSI). Although the number and overall arrangement of these chlorophylls in PSII and PSI differ, structural comparison reveals a cluster of 26 conserved chlorophylls in nearly identical positions and orientations. To explore the role of these conserved chlorophylls within PSII and PSI we studied the influence of their orientation on the efficiency of photochemistry in computer simulations. We found that the native orientations of the conserved chlorophylls were not optimal for light harvesting in either photosystem. However, PSII and PSI each contain two highly orientationally optimized antenna chlorophylls, located close to their respective reaction centers, in positions unique to each photosystem. In both photosystems the orientation of these optimized bridging chlorophylls had a much larger impact on photochemical efficiency than the orientation of any of the conserved chlorophylls. The differential optimization of antenna chlorophyll is discussed in the context of competing selection pressures for the evolution of light harvesting in photosynthesis.

The effect of internal voids in membrane proteins: high-pressure study of two photochemical reaction centres from Rhodobacter sphaeroides

The effect of application of high pressure on the carotenoid-containing bacterial reaction centre from Rhodobacter sphaeroides strain 2.4.1 was studied, and compared to recent experiments performed on its carotenoid-less counterpart, isolated from strain R26.1. Our results indicate that the cavity created by the absence of carotenoid contributes to localised differences in protein compressibility when using the intrinsic chromophores as molecular probes. Differential stability of the electronic transitions of the primary electron donor under high hydrostatic pressure is observed, dependent on the presence of the carotenoid cofactor. This suggests that the transition intensity loss is induced by a slight change of the primary electron donor structure, allowed by the void created by the absence of the carotenoid molecule.

The structure and function of bacterial light-harvesting complexes.

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.

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is approximately 94 A in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 A, and it sits 21 A above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 A. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.

Energy transfer in a single self-aggregated photosynthetic unit

The primary events of bacterial photosynthesis rely on the interplay of various specialized protein complexes organized in a supramolecular structure commonly termed the photosynthetic unit (PSU), which consists of the photochemical reaction center and of an associated antenna network. Employing single-molecule spectroscopic techniques we have been able to observe the excitation-energy transfer within a single PSU. From these findings we conclude that the building blocks of the PSU spontaneously form stable, functional aggregates in a non-membrane environment.

Dynamics of β-Carotene-to-Chlorophyll Singlet Energy Transfer in the Core of Photosystem II

The core of photosystem II comprises three major subcomplexes: the photochemical reaction center (RC) and the core antennas CP43 and CP47. All contain β-carotene as the only carotenoid, but the quantum efficiency of β-carotene-to-chlorophyll singlet energy transfer in the CP43, CP47, and RC complexes is poor. From a femtosecond transient absorption study of the isolated subcomplexes following β-carotene excitation, we show that mainly the β-carotene S2 (1Bu+) state is involved in the energy-transfer process. At most, a minor amount of energy transfer from “hot” β-carotene S1 (2Ag-) states is present in CP43 and CP47, but this S1 contribution is absent in the RC. In all complexes, the relaxed S1 state is not participating in energy transfer. This absence of energy transfer from S1 states explains the low overall yield of β-carotene-to-chlorophyll singlet energy transfer in the PSII core.

Photoinhibition - a historical perspective.

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II 'life cycle.'

Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin-containing enzymes.

Dimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdopterin-containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron-sulphur clusters. Analysis of the N-terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec-dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo-trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.

Membrane Proteins in Bulk Solution Can Be Used for Quasi-Elastic Neutron Scattering Studies: The Case for the Photochemical Reaction Center

The potential to measure the internal dynamics of a membrane protein, the bacterial photochemical reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, was investigated by incoherent quasi-elastic neutron scattering (IQENS) and neutron spin−echo spectroscopy. To isolate and maximize the scattering intensity attributed to the protein, selective deuteration was employed. This deuteration was achieved by embedding detergent-purified RC proteins in a solution containing perdeuterated D28-n-octyl-β-d-glucopyranoside, a sugar−hydroxy detergent. Therefore, the IQENS signal essentially arises only from the motions, which are resolved in time, of the nonexchangeable hydrogen atoms of the protein. This work has established that with a suitable biochemical protocol detailed measurements on the internal dynamics of any integral membrane protein can be undertaken.

Pathways for Energy Transfer in the Core Light-Harvesting Complexes CP43 and CP47 of Photosystem II

The pigment-protein complexes CP43 and CP47 transfer excitation energy from the peripheral antenna of photosystem II toward the photochemical reaction center. We measured the excitation dynamics of the chlorophylls in isolated CP43 and CP47 complexes at 77 K by time-resolved absorbance-difference and fluorescence spectroscopy. The spectral relaxation appeared to occur with rates of 0.2–0.4 ps and 2–3 ps in both complexes, whereas an additional relaxation of 17 ps was observed only in CP47. Using the 3.8-Å crystal structure of the photosystem II core complex from Synechococcus elongatus (A. Zouni, H.-T. Witt, J. Kern, P. Fromme, N. Krauss, W. Saenger, and P. Orth, 2001, Nature, 409:739–743), excitation energy transfer kinetics were calculated and a Monte Carlo simulation of the absorption spectra was performed. In both complexes, the rate of 0.2–0.4 ps can be ascribed to excitation energy transfer within a layer of chlorophylls near the stromal side of the membrane, and the slower 2–3-ps process to excitation energy transfer to the calculated lowest excitonic state. We conclude that excitation energy transfer within CP43 and CP47 is fast and does not contribute significantly to the well-known slow trapping of excitation energy in photosystem II.

The structure and function of CP47 and CP43 in Photosystem II.

This Minireview presents a summary of recent investigations examining the structure and functions of the Photosystem II chlorophyll-proteins CP47 and CP43, updating our previous review which appeared in 1990 (TM Bricker, Photosynth Res 24: 1-13). Since this time, numerous studies have clarified the roles of these chlorophyll-proteins within the photosystem. Biochemical, molecular and structural studies (electron and X-ray diffraction) have demonstrated the close association of these components with the photochemical reaction center of the photosystem and with the extrinsic oxygen evolution enhancer proteins.

Optical Spectroscopy of Individual Photosynthetic Pigment Protein Complexes

Photosynthesis is the process by which plants, algae and photosynthetic bacteria convert solar energy into a form that can be used to sustain the life process. The light reactions occur in closely coupled pigment systems. The energy is absorbed by a network of antenna pigment proteins and efficiently transferred to the photochemical reaction centre where a charge separation takes place providing the free energy for subsequent chemical reactions. The total conversion process, starting with the absorption of a photon and ending with a stable charge separated state occurs within less than 50 ps and has an overall quantum yield of more than 90%. The success of this natural process is based on both the highly efficient absorption of photons by the light-harvesting antenna system and the rapid and efficient transfer of excitation energy to the reaction centre. It is known that most photosynthetic purple bacteria contain two types of antenna complexes, light-harvesting complex 1 (LH1) and light harvesting complex 2 (LH2) which both have a ring-like structure [1,2]. (Some bacterial species like Rhodopseudomonas acidophila contain a third light-harvesting complex termed B800-820.) The reaction centre (RC) presumably forms the core of the LH1 complex, while LH2 complexes are arranged around the perimeter of the LH1 ring in a two-dimensional structure. However the full three-dimensional structure of the whole photosynthetic unit is as yet unknown. The absorption of a photon (mainly) takes place in the LH2 pigments followed by a fast transfer of the excitation energy to the LH1 complex and subsequently to the reaction centre. It appears that the whole structure is highly optimized for capturing light energy and to funnel it to the reaction centre [3-7].

Excited-state dynamics in photosystem II: insights from the x-ray crystal structure.

The heart of oxygenic photosynthesis is photosystem II (PSII), a multisubunit protein complex that uses solar energy to drive the splitting of water and production of molecular oxygen. The effectiveness of the photochemical reaction center of PSII depends on the efficient transfer of excitation energy from the surrounding antenna chlorophylls. A kinetic model for PSII, based on the x-ray crystal structure coordinates of 37 antenna and reaction center pigment molecules, allows us to map the major energy transfer routes from the antenna chlorophylls to the reaction center chromophores. The model shows that energy transfer to the reaction center is slow compared with the rate of primary electron transport and depends on a few bridging chlorophyll molecules. This unexpected energetic isolation of the reaction center in PSII is similar to that found in the bacterial photosystem, conflicts with the established view of the photophysics of PSII, and may be a functional requirement for primary photochemistry in photosynthesis. In addition, the model predicts a value for the intrinsic photochemical rate constant that is 4 times that found in bacterial reaction centers.

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides.

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.