Photochemical Reaction Center Research Articles

Sometimes, red light means grow

Photosynthesis Some cyanobacteria are able to use the far-red end of the light spectrum by synthesizing chlorophyll f pigments. Introducing the protein responsible for chlorophyll f synthesis into crop plants could potentially expand the range of wavelengths that such plants use during photosynthesis and thereby increase their growth efficiency. Ho et al. identified chlorophyll f synthase (ChlF) in two cyanobacteria that are acclimatized to grow using far-red light. Introducing the ChlF-encoding gene into a model cyanobacterium allowed the organism to synthesize chlorophyll f. Similarities between ChlF and a core protein of photosystem II suggest that they have a close evolutionary relationship, and ChlF may even represent a more primitive photochemical reaction center. Science , this issue p. [886][1] [1]: http://www.sciencemag.org/content/353/6302/886.full

Light-dependent chlorophyll f synthase is a highly divergent paralog of PsbA of photosystem II.

Chlorophyll f (Chl f) permits some cyanobacteria to expand the spectral range for photosynthesis by absorbing far-red light. We used reverse genetics and heterologous expression to identify the enzyme for Chl f synthesis. Null mutants of "super-rogue" psbA4 genes, divergent paralogs of psbA genes encoding the D1 core subunit of photosystem II, abolished Chl f synthesis in two cyanobacteria that grow in far-red light. Heterologous expression of the psbA4 gene, which we rename chlF, enables Chl f biosynthesis in Synechococcus sp. PCC 7002. Because the reaction requires light, Chl f synthase is probably a photo-oxidoreductase that employs catalytically useful Chl a molecules, tyrosine YZ, and plastoquinone (as does photosystem II) but lacks a Mn4Ca1O5 cluster. Introduction of Chl f biosynthesis into crop plants could expand their ability to use solar energy.

Photoactivation: The Light-Driven Assembly of the Water Oxidation Complex of Photosystem II.

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. The assembly of the Mn4O5Ca requires light and involves a sequential process called photoactivation. This process harnesses the charge-separation of the photochemical reaction center and the coordination environment provided by the amino acid side chains of the protein to oxidize and organize the incoming manganese ions to form the oxo-bridged metal cluster capable of H2O-oxidation. Although most aspects of this assembly process remain poorly understood, recent advances in the elucidation of the crystal structure of the fully assembled cyanobacterial PSII complex help in the interpretation of the rich history of experiments designed to understand this process. Moreover, recent insights on the structure and stability of the constituent ions of the Mn4CaO5 cluster may guide future experiments. Here we consider the literature and suggest possible models of assembly including one involving single Mn2+ oxidation site for all Mn but requiring ion relocation.

Design principles of natural light-harvesting as revealed by single molecule spectroscopy

Biology offers a boundless source of adaptation, innovation, and inspiration. A wide range of photosynthetic organisms exist that are capable of harvesting solar light in an exceptionally efficient way, using abundant and low-cost materials. These natural light-harvesting complexes consist of proteins that strongly bind a high density of chromophores to capture solar photons and rapidly transfer the excitation energy to the photochemical reaction centre. The amount of harvested light is also delicately tuned to the level of solar radiation to maintain a constant energy throughput at the reaction centre and avoid the accumulation of the products of charge separation. In this Review, recent developments in the understanding of light-harvesting by plants will be discussed, based on results obtained from single molecule spectroscopy studies. Three design principles of the main light-harvesting antenna of plants will be highlighted: (a) fine, photoactive control over the intrinsic protein disorder to efficiently use intrinsically available thermal energy dissipation mechanisms; (b) the design of the protein microenvironment of a low-energy chromophore dimer to control the amount of shade absorption; (c) the design of the exciton manifold to ensure efficient funneling of the harvested light to the terminal emitter cluster.

Colin A. Wraight, 1945-2014.

Colin Allen Wraight, a central figure in photosynthetic electron transfer research since the 1970s, died in Urbana, Illinois, on July 10, 2014. Born in London, England, on November 27, 1945, he had only recently retired from his position as a Professor in Biochemistry, Biophysics & Quantitative Biology, and Plant Biology at the University of Illinois at Urbana-Champaign. Wraight was known especially for his pioneering studies on electron and proton transfer in the photochemical reaction center, and for his careful quantitation of the remarkable quantum efficiency of this device.

Characterization of the structural changes and photochemical activity of photosystem I under Al3+ effect

The photochemical activity of photosystem I (PSI) as affected by Al3+ was investigated in thylakoid membranes and PSI submembrane fractions isolated from spinach. Biophysical and biochemical techniques such as oxygen uptake, light induced absorbance changes at 820nm, chlorophyll fluorescence emission, SDS–polyacrylamide gel electrophoresis, and FTIR spectroscopy have been used to analyze the sites and action modes of this cation on the PSI complex. Our results showed that Al3+ above 3mM induces changes in the redox state of P700 reflected by an increase of P700 photooxidation phase and a delay of the slower rate of P700 re-reduction which reveals that Al3+ exerted an inhibitory action at the donor side of PSI especially at plastocyanin (PC). Furthermore, results of P700 photooxidation monitored in the presence of DCMU with or without MV suggested that the same range of Al3+ concentrations impairs the photochemical reaction centers (RC) of PSI, as shown by the decline in the amount of active population of P700, and disrupts the charge separation between P700 and the primary electron acceptor A0 leading to the inhibition of electron transfer at the acceptor side of PSI. These inhibitory actions were also accompanied by an impairment of the energy transfer from light harvesting complex (LHCI) to RC of PSI, following the disconnection of LHCI antenna as illustrated by an enhancement of chlorophyll fluorescence emission spectra at low temperature (77K). The above results coincided with FTIR measurements that indicated a conformational change of the protein secondary structures in PSI complex where 25% of α-helix was converted into β-sheet, β-antiparallel and turn structures. These structural changes in PSI complex proteins are closely related with the alteration photochemical activity of PSI including the inhibition of the electron transport through both acceptor and donor sides of PSI.

A fresh look at the evolution and diversification of photochemical reaction centers.

In this review, I reexamine the origin and diversification of photochemical reaction centers based on the known phylogenetic relations of the core subunits, and with the aid of sequence and structural alignments. I show, for example, that the protein folds at the C-terminus of the D1 and D2 subunits of Photosystem II, which are essential for the coordination of the water-oxidizing complex, were already in place in the most ancestral Type II reaction center subunit. I then evaluate the evolution of reaction centers in the context of the rise and expansion of the different groups of bacteria based on recent large-scale phylogenetic analyses. I find that the Heliobacteriaceae family of Firmicutes appears to be the earliest branching of the known groups of phototrophic bacteria; however, the origin of photochemical reaction centers and chlorophyll synthesis cannot be placed in this group. Moreover, it becomes evident that the Acidobacteria and the Proteobacteria shared a more recent common phototrophic ancestor, and this is also likely for the Chloroflexi and the Cyanobacteria. Finally, I argue that the discrepancies among the phylogenies of the reaction center proteins, chlorophyll synthesis enzymes, and the species tree of bacteria are best explained if both types of photochemical reaction centers evolved before the diversification of the known phyla of phototrophic bacteria. The primordial phototrophic ancestor must have had both Type I and Type II reaction centers.

Modulating energy arriving at photochemical reaction centers: orange carotenoid protein-related photoprotection and state transitions.

Photosynthetic organisms tightly regulate the energy arriving to the reaction centers in order to avoid photodamage or imbalance between the photosystems. To this purpose, cyanobacteria have developed mechanisms involving relatively rapid (seconds to minutes) changes in the photosynthetic apparatus. In this review, two of these processes will be described: orange carotenoid protein(OCP)-related photoprotection and state transitions which optimize energy distribution between the two photosystems. The photoactive OCP is a light intensity sensor and an energy dissipater. Photoactivation depends on light intensity and only the red-active OCP form, by interacting with phycobilisome cores, increases thermal energy dissipation at the level of the antenna. A second protein, the "fluorescence recovery protein", is needed to recover full antenna capacity under low light conditions. This protein accelerates OCP conversion to the inactive orange form and plays a role in dislodging the red OCP protein from the phycobilisome. The mechanism of state transitions is still controversial. Changes in the redox state of the plastoquinone pool induce movement of phycobilisomes and/or photosystems leading to redistribution of energy absorbed by phycobilisomes between PSII and PSI and/or to changes in excitation energy spillover between photosystems. The different steps going from the induction of redox changes to movement of phycobilisomes or photosystems remain to be elucidated.

Enhanced biofuel production using optimality, pathway modification and waste minimization

In response to their environment, algae in the wild may use an approximation to optimality of resource allocation in cellular structures, photosynthetic pigments, enzymes, transporters in membranes and RNAs and in their genetic material. However, under controlled conditions, when algae are grown for biofuel (lipid) production for example, some of these processes can be altered to increase the efficiency of photosynthesis and therefore, lipid yield. This suggests that there is scope for selecting mutations and for genetic engineering at various levels in the photosynthetic apparatus with the aim of increasing efficiency of photon use and the rate of transformation of resources per unit biomass to improve biofuel yields. More specifically, the wavelength range covered by photosynthetic pigments and photochemical reaction centres could be increased, the number of protons transported from the thylakoid lumen to the stroma per unit ATP synthesised by the ATP synthetase could be decreased, the fluctuating light effect could be utilised and photosynthetic pathways changed, e.g. replacing part or all of the current machinery for autotrophic fixation of inorganic carbon. There are also possibilities for decreasing carbon loss by decreasing ‘wasteful’ aspects of dark respiration and of dissolved organic carbon loss. Provided that the environmental fluctuations to which algal growth conditions are constrained, there are possibilities for decreasing the resource costs of protection from ROS, and by down-regulating photoprotective mechanisms, as well as limiting the capacity to repair processes related to photoinhibition. Decreased protein turnover is also a potential energetic saving. These interventions apply to individual processes; however, this may not be immediately incorporated into the optimal allocation of resources by the alga, and further intervention using a system biology approach may be required.

Variation in supramolecular organisation of the photosynthetic membrane of Rhodobacter sphaeroides induced by alteration of PufX

In purple bacteria of the genus Rhodobacter (Rba.), an LH1 antenna complex surrounds the photochemical reaction centre (RC) with a PufX protein preventing the LH1 complex from completely encircling the RC. In membranes of Rba. sphaeroides, RC-LH1 complexes associate as dimers which in turn assemble into longer range ordered arrays. The present work uses linear dichroism (LD) and dark-minus-light difference LD (ΔLD) to probe the organisation of genetically altered RC-LH1 complexes in intact membranes. The data support previous proposals that Rba. capsulatus, and Rba. sphaeroides heterologously expressing the PufX protein from Rba. capsulatus, produce monomeric core complexes in membranes that lack long-range order. Similarly, Rba. sphaeroides with a point mutation in the Gly 51 residue of PufX, which is located on the membrane-periplasm interface, assembles mainly non-ordered RC-LH1 complexes that are most likely monomeric. All the Rba. sphaeroides membranes in their ΔLD spectra exhibited a spectral fingerprint of small degree of organisation implying the possibility of ordering influence of LH1, and leading to an important conclusion that PufX itself has no influence on ordering RC-LH1 complexes, as long-range order appears to be induced only through its role of configuring RC-LH1 complexes into dimers.

Functional Interfacing of Rhodospirillum rubrum Chromatophores to a Conducting Support for Capture and Conversion of Solar Energy

Owing to the considerable current interest in replacing fossil fuels with solar radiation as a clean, renewable, and secure energy source, light-driven electron transport in natural photosynthetic systems offers a valuable blueprint for conversion of sunlight to useful energy forms. In particular, intracytoplasmic membrane vesicles (chromatophores) from the purple bacterium Rhodospirillum rubrum provide a fully functional and robust photosynthetic apparatus, ideal for biophysical investigations of energy transduction and incorporation into biohybrid photoelectrochemical devices. These vesicular organelles, which arise by invagination of the cytoplasmic membrane, are the sites of the photochemical reaction centers and the light harvesting 1 (LH1) complex. The LH1 protein is responsible for collecting visible and near-IR radiant energy and funneling these excitations to the reaction center for conversion into a transmembrane charge separation. Here, we have investigated the morphology, fluorescence kinetics and photocurrent generation of chromatophores from Rsp. rubrum deposited directly onto gold surfaces in the absence of chemical surface modifications. Atomic force microscopy showed a significant coverage of the gold electrode surface by Rsp. rubrum chromatophores. By in situ fluorescence induction/relaxation measurements, a high retention of the quantum yield of photochemistry was demonstrated in the photoactive films. Chronoamperometric measurements showed that the assembled bioelectrodes were capable of generating sustained photocurrent under white light illumination at 220 mW/cm(2) with a maximum current of 1.5 μA/cm(2), which slowly declines in about 1 week. This study demonstrates the possibility of photoelectrochemical control of robust chromatophore preparations from Rsp. rubrum that paves the way for future incorporation into functional solar cells.

Spectral expansion and antenna reduction can enhance photosynthesis for energy production

We consider two approaches for improving the efficiency of photosynthesis: the expansion of the solar spectrum that drives oxygenic photosynthesis and the reduction of antenna systems that couple to the photochemical reaction center. The first approach can possibly result in an increase of 19% in the number of photons available per unit area if the photosynthetically active radiation spectrum is expanded to 750 nm. The second approach can in principle shift the photosynthesis light response curve to significantly higher intensities, thereby reducing the amount of excess light, which is absorbed photons in excess of the number that can be utilized. The implementation of these approaches may lead to apparent improvement in photosynthetic efficiency in many but not all the cases.

Global abundance of microbial rhodopsins

Photochemical reaction centers and rhodopsins are the only phototrophic mechanisms known to have evolved on Earth. The minimal cost of bearing a rhodopsin-based phototrophic mechanism in comparison to maintaining a photochemical reaction center suggests that rhodopsin is the more abundant of the two. We tested this hypothesis by conducting a global abundance calculation of phototrophic mechanisms from 116 marine and terrestrial microbial metagenomes. On average, 48% of the cells from which these metagenomes were generated harbored a rhodopsin gene, exceeding the reaction center abundance by threefold. Evidence from metatranscriptomic data suggests that this genomic potential is realized to a substantial extent, at least for the small-sized (>0.8 μm) of microbial fractions.

Gain and Loss of Phototrophic Genes Revealed by Comparison of Two Citromicrobium Bacterial Genomes

Proteobacteria are thought to have diverged from a phototrophic ancestor, according to the scattered distribution of phototrophy throughout the proteobacterial clade, and so the occurrence of numerous closely related phototrophic and chemotrophic microorganisms may be the result of the loss of genes for phototrophy. A widespread form of bacterial phototrophy is based on the photochemical reaction center, encoded by puf and puh operons that typically are in a ‘photosynthesis gene cluster’ (abbreviated as the PGC) with pigment biosynthesis genes. Comparison of two closely related Citromicrobial genomes (98.1% sequence identity of complete 16S rRNA genes), Citromicrobium sp. JL354, which contains two copies of reaction center genes, and Citromicrobium strain JLT1363, which is chemotrophic, revealed evidence for the loss of phototrophic genes. However, evidence of horizontal gene transfer was found in these two bacterial genomes. An incomplete PGC (pufLMC-puhCBA) in strain JL354 was located within an integrating conjugative element, which indicates a potential mechanism for the horizontal transfer of genes for phototrophy.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c₂ and K₄[Fe(CN)₆]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

Diversity of Purple Phototrophic Bacteria, Inferred from <i>pufM</i> Gene, within Epilithic Biofilm in Tama River, Japan

The diversity of purple phototrophic bacteria in algae-dominated biofilm of a streambed in Tama River, Japan was investigated. Clone library analysis of the pufM gene encoding a subunit of the photochemical reaction center of purple bacteria detected 18 operational taxonomic units (OTUs) in several classes of Proteobacteria. Most of the OTUs showed less than 85% identity to the PufM amino acid sequences of known phototrophic bacteria. These results suggest that phylogenetically divergent and unknown purple phototrophic bacteria are present in the epilithic biofilm of the river.

Synthesis and Characterization of Nonsymmetric Cyclopentene-Based Dithienylethenes

Nonsymmetric cyclopentene-based dithienylethenes, containing both thienyl and benzothienyl units, have been synthesized for the first time, employing intramolecular McMurry reaction as a key transformation to target compounds 10 and 16. Photochromic properties of these compounds were examined in toluene and acetonitrile solutions, PMMA layers, solid films, and crystal phase. To explain an unprecedented single-crystalline photochromism phenomena of 10, X-ray crystallographic analysis was performed, revealing a significant influence of strong S···S interatomic interactions on the intramolecular distance between two photochemical reaction centers in the molecule. Compound 16 could be further synthetically modified in a step-by-step manner, thus serving as potential key intermediate to various complex bifunctional photochromic molecules.

Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC–LH1 core complex

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.

Effects of the measuring light on the photochemistry of the bacterial photosynthetic reaction center from Rhodobacter sphaeroides

The bacterial reaction center (RC) has become a reference model in the study of the diverse interactions of quinones with electron transfer complexes. In these studies, the RC functionality was probed through flash-induced absorption changes where the state of the primary donor is probed by means of a continuous measuring beam and the electron transfer is triggered by a short intense light pulse. The single-beam set-up implies the use as reference of the transmittance measured before the light pulse. Implicit in the analysis of these data is the assumption that the measuring beam does not elicit the protein photochemistry. At variance, measuring beam is actinic in nature at almost all the suitable wavelengths. In this contribution, the analytical modelling of the time evolution of neutral and charge-separated RCs has been performed. The ability of measuring light to elicit RC photochemistry induces a first order growth of the charge-separated state up to a steady state that depends on the light intensity and on the occupation of the secondary quinone (Q(B)) site. Then the laser pulse pumps all the RCs in the charge-separated state. The following charge recombination is still affected by the measuring beam. Actually, the kinetics of charge recombination measured in RC preparation with the Q(B) site partially occupied are two-exponential. The rate constant of both fast and slow phases depends linearly on the intensity of the measuring beam while their relative weights depend not only on the fractions of RC with the Q(B) site occupied but also on the measuring light intensity itself.

Identification of a Gene Essential for the First Committed Step in the Biosynthesis of Bacteriochlorophyll c

Bacteriochlorophylls (BChls) c, d, and e are the major chlorophylls in chlorosomes, which are the largest and one of the most efficient antennae produced by chlorophototrophic organisms. In the biosynthesis of these three BChls, a C-13(2)-methylcarboxyl group found in all other chlorophylls (Chls) must be removed. This reaction is postulated to be the first committed step in the synthesis of these BChls. Analyses of gene neighborhoods of (B)Chl biosynthesis genes and distribution patterns in organisms producing chlorosomes helped to identify a gene (bciC) that appeared to be a good candidate to produce the enzyme involved in this biochemical reaction. To confirm that this was the case, a deletion mutant of an open reading frame orthologous to bciC, CT1077, was constructed in Chlorobaculum tepidum, a genetically tractible green sulfur bacterium. The CT1077 deletion mutant was unable to synthesize BChl c but still synthesized BChl a and Chl a. The deletion mutant accumulated large amounts of various (bacterio)pheophorbides, all of which still had C-13(2)-methylcarboxyl groups. A C. tepidum strain in which CT1077 was replaced by an orthologous gene, Cabther_B0081 [corrected] from "Candidatus Chloracidobacterium thermophilum" was constructed. Although the product of Cabther_B0081 [corrected] was only 28% identical to the product of CT1077, this strain synthesized BChl c, BChl a, and Chl a in amounts similar to wild-type C. tepidum cells. To indicate their roles in the first committed step of BChl c, d, and e biosynthesis, open reading frames CT1077 and Cabther_B0081 [corrected] have been redesignated bciC. The potential mechanism by which BciC removes the C-13(2)-methylcarboxyl moiety of chlorophyllide a is discussed.