Biomedical subjects

Robert E Blankenship

Publications and source records attributed to Robert E Blankenship.

At least 19 recordsLinked to original sources

PubMed2006

Variable fluorescence in green sulfur bacteria.

Green sulfur bacteria possess a complex photosynthetic machinery. The dominant light harvesting systems are chlorosomes, which consist of bacteriochlorophyll c, d or e oligomers with small amounts of protein. The chlorosomes are energetically coupled to the membrane-embedded iron sulfur-type reaction center via a bacteriochlorophyll a-containing baseplate protein and the Fenna-Matthews-Olson (FMO) antenna protein. The fluorescence yield and spectral properties of these photosynthetic complexes were investigated in intact cells of several species of green sulfur bacteria under physiological, anaerobic conditions. Surprisingly, green sulfur bacteria show a complex modulation of fluorescence yield upon illumination that is very similar to that observed in oxygenic phototrophs. Within a few seconds of illumination, the fluorescence reaches a maximum, which decreases within a minute of illumination to a lower steady state. Fluorescence spectroscopy reveals that the fluorescence yield during both processes is primarily modulated on the FMO-protein level, while the emission from chlorosomes remains mostly unchanged. The two most likely candidates that modulate bacteriochlorophyll fluorescence are (1) direct excitation quenching at the FMO-protein level and (2) indirect modulation of FMO-protein fluorescence by the reduction state of electron carriers that are part of the reaction center.

Bacterial Proteins↗

PubMed2006

The complete genome sequence of Roseobacter denitrificans reveals a mixotrophic rather than photosynthetic metabolism.

Purple aerobic anoxygenic phototrophs (AAPs) are the only organisms known to capture light energy to enhance growth only in the presence of oxygen but do not produce oxygen. The highly adaptive AAPs compose more than 10% of the microbial community in some euphotic upper ocean waters and are potentially major contributors to the fixation of the greenhouse gas CO2. We present the complete genomic sequence and feature analysis of the AAP Roseobacter denitrificans, which reveal clues to its physiology. The genome lacks genes that code for known photosynthetic carbon fixation pathways, and most notably missing are genes for the Calvin cycle enzymes ribulose bisphosphate carboxylase (RuBisCO) and phosphoribulokinase. Phylogenetic evidence implies that this absence could be due to a gene loss from a RuBisCO-containing alpha-proteobacterial ancestor. We describe the potential importance of mixotrophic rather than autotrophic CO2 fixation pathways in these organisms and suggest that these pathways function to fix CO2 for the formation of cellular components but do not permit autotrophic growth. While some genes that code for the redox-dependent regulation of photosynthetic machinery are present, many light sensors and transcriptional regulatory motifs found in purple photosynthetic bacteria are absent.

Amino Acid Sequence↗

PubMed2006

Conservation of distantly related membrane proteins: photosynthetic reaction centers share a common structural core.

Photosynthesis was established on Earth more than 3 billion years ago. All available evidences suggest that the earliest photosynthetic organisms were anoxygenic and that oxygen-evolving photosynthesis is a more recent development. The reaction center complexes that form the heart of the energy storage process are integral membrane pigment proteins that span the membrane in vectorial fashion to carry out electron transfer. The origin and extent of distribution of these proteins has been perplexing from a phylogenetic point of view mostly because of extreme sequence divergence. A series of integral membrane proteins of known structure and varying degrees of sequence identity have been compared using combinatorial extension-Monte Carlo methods. The proteins include photosynthetic reaction centers from proteobacteria and cyanobacterial photosystems I and II, as well as cytochrome oxidase, bacteriorhodopsin, and cytochrome b. The reaction center complexes show a remarkable conservation of the core structure of 5 transmembrane helices, strongly implying common ancestry, even though the residual sequence identity is less than 10%, whereas the other proteins have structures that are unrelated. A relationship of sequence with structure was derived from the reaction center structures; with characteristic decay length of 1.6 A. Phylogenetic trees derived from the structural alignments give insights into the earliest photosynthetic reaction center, strongly suggesting that it was a homodimeric complex that did not evolve oxygen.

Amino Acid Sequence↗

PubMed2006

Light harvesting in photosystem I supercomplexes.

In photosynthetic membranes of cyanobacteria, algae, and higher plants, photosystem I (PSI) mediates light-driven transmembrane electron transfer from plastocyanin or cytochrome c6 to the ferredoxin-NADP complex. The oxidoreductase function of PSI is sensitized by a reversible photooxidation of primary electron donor P700, which launches a multistep electron transfer via a series of redox cofactors of the reaction center (RC). The excitation energy for the functioning of the primary electron donor in the RC is delivered via the chlorophyll core antenna in the complex with peripheral light-harvesting antennas. Supermolecular complexes of the PSI acquire remarkably different structural forms of the peripheral light-harvesting antenna complexes, including distinct pigment types and organizational principles. The PSI core antenna, being the main functional unit of the supercomplexes, provides an increased functional connectivity in the chlorophyll antenna network due to dense pigment packing resulting in a fast spread of the excitation among the neighbors. Functional connectivity within the network as well as the spectral overlap of antenna pigments allows equilibration of the excitation energy in the depth of the whole membrane within picoseconds and loss-free delivery of the excitation to primary donor P700 within 20-40 ps. Low-light-adapted cyanobacteria under iron-deficiency conditions extend this capacity via assembly of efficiently energy coupled rings of CP43-like complexes around the PSI trimers. In green algae and higher plants, less efficient energy coupling in the eukaryotic PSI-LHCI supercomplexes is probably a result of the structural adaptation of the Chl a/b binding LHCI peripheral antenna that not only extends the absorption cross section of the PSI core but participates in regulation of excitation flows between the two photosystems as well as in photoprotection.

Light-Harvesting Protein Complexes↗

PubMed2005

The nature of the photosystem II reaction centre in the chlorophyll d-containing prokaryote, Acaryochloris marina.

Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.

Chlorophyll↗

PubMed2005

New class of bacterial membrane oxidoreductases.

A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purified from the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus, and putative operons for similar complexes were identified in a wide range of bacteria. In most cases, the presence of the new complex is anticorrelated with the cytochrome bc or bf electron-transfer complex, suggesting that it replaces it functionally. This appears to be a widespread yet previously unrecognized protein complex involved in energy metabolism in bacteria.

Amino Acid Sequence↗

PubMed2005

An obligately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent.

The abundance of life on Earth is almost entirely due to biological photosynthesis, which depends on light energy. The source of light in natural habitats has heretofore been thought to be the sun, thus restricting photosynthesis to solar photic environments on the surface of the Earth. If photosynthesis could take place in geothermally illuminated environments, it would increase the diversity of photosynthetic habitats both on Earth and on other worlds that have been proposed to possibly harbor life. Green sulfur bacteria are anaerobes that require light for growth by the oxidation of sulfur compounds to reduce CO2 to organic carbon, and are capable of photosynthetic growth at extremely low light intensities. We describe the isolation and cultivation of a previously unknown green sulfur bacterial species from a deep-sea hydrothermal vent, where the only source of light is geothermal radiation that includes wavelengths absorbed by photosynthetic pigments of this organism.

Bacteria, Anaerobic↗

PubMed2005

Digital position determination system for electron microscopy.

The precise determination of object positions within a specimen grid is important for many applications in electron microscopy. For example, real-time position determination is necessary for current statistical approaches and the efficient mapping and relocation of objects. Unfortunately, precise real-time position determination is not available on many older electron microscopes with manual stage controls. This report demonstrates the cost-effective and flexible implementation of a digital position determination system that can be adapted to many hand-operated electron microscopes. A customized solution that includes the hardware and software to accomplish position determination is presented. Lists of required parts, instructions for building the hardware, and descriptions of the developed programs are included. Two LED-photodiode assemblies detect x and y movements via an optical wheel that is in physical contact with the mechanical x and y stage control elements. These detector assemblies are interfaced with an integrated circuit that converts movement information into serial port-compatible signals, which are interpreted by a computer with specialized software. Two electron microscopes, a Philips CM12 (S)TEM and a Philips 201 TEM, were equipped with the described digital position determination system. The position fidelity and position fidelity after reloading of grids were determined for both microscopes. The determined position deviation was 1.06 microm in the x axis and 0.565 microm in the y axis for the Philips CM12 (S)TEM, and 0.303 microm in the x axis and 0.545 microm in the y axis for the Philips 201 TEM. After reloading and computational realigning, the determined average position variation was 2.66 microm in the x axis and 2.61 microm in the y axis for the Philips CM12 (S)TEM, and 1.13 microm in the x axis and 1.27 microm in the y axis for the Philips 201 TEM.

Electronics↗

PubMed2005

Two-dimensional spectroscopy of electronic couplings in photosynthesis.

Time-resolved optical spectroscopy is widely used to study vibrational and electronic dynamics by monitoring transient changes in excited state populations on a femtosecond timescale. Yet the fundamental cause of electronic and vibrational dynamics--the coupling between the different energy levels involved--is usually inferred only indirectly. Two-dimensional femtosecond infrared spectroscopy based on the heterodyne detection of three-pulse photon echoes has recently allowed the direct mapping of vibrational couplings, yielding transient structural information. Here we extend the approach to the visible range and directly measure electronic couplings in a molecular complex, the Fenna-Matthews-Olson photosynthetic light-harvesting protein. As in all photosynthetic systems, the conversion of light into chemical energy is driven by electronic couplings that ensure the efficient transport of energy from light-capturing antenna pigments to the reaction centre. We monitor this process as a function of time and frequency and show that excitation energy does not simply cascade stepwise down the energy ladder. We find instead distinct energy transport pathways that depend sensitively on the detailed spatial properties of the delocalized excited-state wavefunctions of the whole pigment-protein complex.

Bacterial Proteins↗

PubMed2005

Discovery of a free-living chlorophyll d-producing cyanobacterium with a hybrid proteobacterial/cyanobacterial small-subunit rRNA gene.

Chlorophyll d-producing cyanobacteria are a recently described group of phototrophic bacteria that is a major focus of photosynthesis research, previously known only from marine environments in symbiosis with eukaryotes. We have discovered a free-living member of this group from a eutrophic hypersaline lake. Phylogenetic analyses indicated these strains are closely related to each other but not to prochlorophyte cyanobacteria that also use an alternative form of chlorophyll as the major light-harvesting pigment. We have also demonstrated that these bacteria acquired a fragment of the small-subunit rRNA gene encoding a conserved hairpin in the bacterial ribosome from a proteobacterial donor at least 10 million years before the present. Thus, our most widely used phylogenetic marker can be a mosaic of sequence fragments with widely divergent evolutionary histories.

Base Sequence↗

PubMed2005

Structural and functional organization of the peripheral light-harvesting system in photosystem I.

This review centers on the structural and functional organization of the light-harvesting system in the peripheral antenna of Photosystem I (LHC I) and its energy coupling to the Photosystem I (PS I) core antenna network in view of recently available structural models of the eukaryotic Photosystem I-LHC I complex, eukaryotic LHC II complexes and the cyanobacterial Photosystem I core. A structural model based on the 3D homology of Lhca4 with LHC II is used for analysis of the principles of pigment arrangement in the LHC I peripheral antenna, for prediction of the protein ligands for the pigments that are unique for LHC I and for estimates of the excitonic coupling in strongly interacting pigment dimers. The presence of chlorophyll clusters with strong pigment-pigment interactions is a structural feature of PS I, resulting in the characteristic red-shifted fluorescence. Analysis of the interactions between the PS I core antenna and the peripheral antenna leads to the suggestion that the specific function of the red pigments is likely to be determined by their localization with respect to the reaction center. In the PS I core antenna, the Chl clusters with a different magnitude of low energy shift contribute to better spectral overlap of Chls in the reaction center and the Chls of the antenna network, concentrate the excitation around the reaction center and participate in downhill enhancement of energy transfer from LHC II to the PS I core. Chlorophyll clusters forming terminal emitters in LHC I are likely to be involved in photoprotection against excess energy.

Amino Acid Sequence↗

PubMed2005

The ultrastructure of Chlorobium tepidum chlorosomes revealed by electron microscopy.

Chlorosomes are the light harvesting structures of green photosynthetic bacteria. Each chlorosome from green sulfur bacteria houses hundreds of thousands of bacteriochlorophyll molecules in addition to smaller amounts of chlorobiumquinone and carotenoids. In electron microscopy studies, chlorosomes exhibit different appearances depending on the fixation method used. Fixation with osmium tetroxide results in electron-transparent chlorosomes. Fixation with potassium permanganate results in clearly delineated electron-dense chlorosomes. This fixation method features an electron-transparent area in the interior of the chlorosome. In addition to electron density patterns that can be considered compositions of rod-shaped elements, chlorosomes exhibit a striation pattern that is oriented parallel to the longitudinal axis. Treatment with osmium tetroxide followed by potassium permanganate treatment results in a more diffused density distribution that outlines connecting elements between the chlorosome and the cytoplasmic membrane, and connecting elements between the cytoplasmic membrane and the outer membrane, which act as a diffusion barrier for electron density.

Cell Membrane↗

PubMed2005

Spectral and kinetic analysis of the energy coupling in the PS I-LHC I supercomplex from the green alga Chlamydomonas reinhardtii at 77 K.

Energy transfer processes in the chlorophyll antenna of the PS I-LHCI supercomplexes from the green alga Chlamydomonas reinhardtii have been studied at 77 K using transient absorption spectroscopy with multicolor excitation in the 640-670 nm region. Comparison of the kinetic data obtained at low and room temperatures indicates that the slow approximately approximately 100 ps excitation equilibration phase that is characteristic of energy coupling of the LHCI peripheral antenna to the PS I core at physiological temperatures (Melkozernov AN, Kargul J, Lin S, Barber J and Blankenship RE (2004) J Phys Chem B 108: 10547-10555) is not observed in the excitation dynamics of the PS I-LHCI supercomplex at 77 K. This suggests that at low temperatures the peripheral antenna is energetically uncoupled from the PS I core antenna. Under these conditions the observed kinetic phases on the time scales from subpicoseconds to tens of picoseconds represent the superposition of the processes occurring independently in the PS I core antenna and the Chl a/b containing LHCI antenna. In the PS I-LHCI supercomplex with two uncoupled antennas the excitation is channeled to the excitation sinks formed at low temperature by clusters of red pigments. A better spectral resolution of the transient absorption spectra at 77 K results in detection of two DeltaA bands originating from the rise of photobleaching on the picosecond time scale of two clearly distinguished pools of low energy absorbing Chls in the PS I-LHCI supercomplex. The first pool of low energy pigments absorbing at 687 nm is likely to originate from the red pigments in the LHCI where the Lhca1 protein is most abundant. The second pool at 697 nm is suggested to result either from the structural interaction of the LHCI and the PS I core or from other Lhca proteins in the antenna. The kinetic data are discussed based on recent structural models of the PS I-LHCI. It is proposed that the uncoupling of pigment pools may be a control mechanism that regulates energy flow in Photosystem I.

Animals↗

PubMed2005

Purification and characterization of the B808-866 light-harvesting complex from green filamentous bacterium Chloroflexus aurantiacus.

The integral membrane light-harvesting complex B808-866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808-866 antenna complex is 36 +/- 2 per reaction center. The main carotenoid type is gamma-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808-866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808-866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808-866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808-866. The unique properties of this light-harvesting complex may provide insights on the protein-pigment interactions in bacterial photosynthesis.

Bacterial Proteins↗

PubMed2005

Effect of iron on growth and ultrastructure of Acaryochloris marina.

The cyanobacterial genus Acaryochloris is the only known group of oxygenic phototrophs that contain chlorophyll d rather than chlorophyll a as the major photosynthetic pigment. Studies on this organism are still in their earliest stages, and biochemical analysis has rapidly outpaced growth optimization. We have investigated culture growth of the major strains of Acaryochloris marina (MBIC11017 and MBIC10697) by using several published and some newly developed growth media. It was determined that heavy addition of iron significantly enhanced culture longevity. These high-iron cultures showed an ultrastructure with thylakoid stacks that resemble traditional cyanobacteria (unlike previous studies). These cultures also show a novel reversal in the pigment ratios of the photosystem II signature components chlorophyll a and pheophytin a, as opposed to those in previous studies.

Chromatography, High Pressure Liquid↗

PubMed2004

The evolutionary development of the protein complement of photosystem 2.

During the transition from anoxygenic to oxygenic photosynthesis, the Type 2 reaction center underwent many changes, none so dramatic as the remarkable increase in complexity at the protein level, from only three or four subunits in the anoxygenic reaction center to possibly more than 25 in Photosystem 2 (PS2). The evolutionary source of most of these proteins is enigmatic, as they have no apparent homology to any other proteins in existing databases. However, some of the proteins in PS2 have apparent homologies to each other, suggesting ancient gene duplications have played an important role in the development of the complex. These homologies include the well-known examples of the D1 and D2 reaction center core proteins and the CP43 and CP47 core antenna proteins. In addition, PsbE and PsbF, the two subunits comprising cytochrome b-559, show homology to each other, suggesting that a homodimeric cytochrome preceded the heterodimeric one. Other potential homologies that appear to be statistically significant include PsbV with the N-terminal part of D1 and PsbT with PsbI. Most of the proteins that make up the photosynthetic apparatus bear no relation to any other proteins from any source. This suggests that a period of remarkable evolutionary innovation took place when the ability to make oxygen was invented. This was probably a response to the production of highly toxic oxygen and these new proteins served to protect and repair the photosynthetic apparatus from the harmful effects of oxygen.

Cyanobacteria↗

PubMed2004

Spectroscopic properties of the main-form and high-salt peridinin-chlorophyll a proteins from Amphidinium carterae.

The main-form (MFPCP) and high-salt (HSPCP) peridinin-chlorophyll a proteins from the dinoflagellate Amphidinium carterae were investigated using absorption, fluorescence, fluorescence excitation, two-photon, and fast-transient optical spectroscopy. Pigment analysis has demonstrated previously that MFPCP contains eight peridinins and two chlorophyll (Chl) a molecules, whereas HSPCP has six peridinins and two Chl a molecules [Sharples, F. P., et al. (1996) Biochim. Biophys. Acta 1276, 117-123]. Absorption spectra of the complexes were recorded at 10 K and analyzed in the 400-600 nm region by summing the individual 10 K spectra of Chl a and peridinin recorded in 2-MTHF. The absorption spectral profiles of the complexes in the Q(y) region between 650 and 700 nm were fit using Gaussian functions. The absorption and fluorescence spectra from both complexes exhibit several distinguishing features that become evident only at cryogenic temperatures. In particular, at low temperatures the Q(y) transitions of the Chls bound in the HSPCP complex are split into two well-resolved bands. Fluorescence excitation spectroscopy has revealed that the peridinin-to-Chl a energy transfer efficiency is high (>95%). Transient absorption spectroscopy has been used to measure the rate of energy transfer between the two bound Chls which is a factor of 2.9 slower in HSPCP than in MFPCP. The kinetic data are interpreted in terms of the Förster mechanism describing energy transfer between weakly coupled, spatially fixed, donor-acceptor Chl a molecules. The study provides insight into the molecular factors that control energy transfer in this class of light-harvesting pigment-protein complexes.

Ammonium Sulfate↗

PubMed2004

Time line of discoveries: anoxygenic bacterial photosynthesis.

A time line of important research relating to anoxygenic photosynthetic organisms is presented. The time line includes discoveries of organisms, metabolic capabilities, molecular complexes and genetic systems. It also pinpoints important milestones in our understanding of the structure, function, organization, assembly and regulation of photosynthetic complexes.

Journal Article↗