The Experts below are selected from a list of 246 Experts worldwide ranked by ideXlab platform
Michael T Madigan - One of the best experts on this subject based on the ideXlab platform.
-
The Chlorobiaceae, Chloroflexaceae , and Heliobacteriaceae
Modern Topics in the Phototrophic Prokaryotes, 2017Co-Authors: Michael T Madigan, Nicole A. Vander Schaaf, W. Matthew SattleyAbstract:Here we examine the basic biology of three major groups of “green” anoxygenic phototrophic bacteria: the green sulfur bacteria (Chlorobiaceae), the green nonsulfur bacteria (also called the filamentous anoxygenic phototrophs) (Chloroflexaceae), and the heliobacteria (Heliobacteriaceae). Only organisms that have been grown in laboratory culture are considered. Interestingly, the model organisms for each family are thermophiles: the green sulfur bacterium Chlorobaculum tepidum, the filamentous green nonsulfur bacterium Chloroflexus aurantiacus, and the hot spring Heliobacterium species, Heliobacterium modesticaldum. All model green bacteria have had their genomes sequenced, and in the green sulfur bacteria, genome sequences of all recognized species have been completed and compared. Although species in each family are distinct from species in each of the other families in many ways, there are key properties that unite two families to the exclusion of the third. These include the presence of chlorosomes in the green sulfur and green nonsulfur bacteria and the structure of the reaction centers in the green sulfur bacteria and heliobacteria. However, the three families of green-colored bacteria are phylogenetically distinct and thus any similarities are likely the result of horizontal gene transfers.
-
Amino acid-assimilating phototrophic heliobacteria from soda lake environments: Heliorestis acidaminivorans sp. nov. and ‘Candidatus Heliomonas lunata’
Extremophiles, 2012Co-Authors: Marie Asao, Shinichi Takaichi, Michael T MadiganAbstract:Two novel taxa of heliobacteria, Heliorestis acidaminivorans sp. nov. strain HR10B^T and ‘ Candidatus Heliomonas lunata’ strain SLH, were cultured from shoreline sediments/soil of Lake El Hamra (Egypt) and lake water/benthic sediments of Soap Lake (USA), respectively; both are highly alkaline soda lakes. Cells of strain HR10B were straight rods, while cells of strain SLH were curved rods. Both organisms were obligate anaerobes, produced bacteriochlorophyll g , and lacked intracytoplasmic photosynthetic membrane systems. Although the absorption spectrum of strain HR10B was typical of other heliobacteria, that of strain SLH showed unusually strong absorbance of the OH-chlorophyll a component. Major carotenoids of both organisms were OH-diaponeurosporene glucosyl esters, as in other alkaliphilic heliobacteria, and both displayed an alkaliphilic and mesophilic phenotype. Strain HR10B was remarkable among heliobacteria in its capacity to photoassimilate a number of carbon sources, including several amino acids. Nitrogenase activity was observed in strain HR10B, but not in strain SLH. The 16S ribosomal RNA gene tree placed strain HR10B within the genus Heliorestis , but distinct from other described species. By contrast, strain SLH was phylogenetically more closely related to neutrophilic heliobacteria and is the first alkaliphilic Heliobacterium known outside of the genus Heliorestis .
-
Taxonomy, phylogeny, and ecology of the heliobacteria
Photosynthesis Research, 2010Co-Authors: Marie Asao, Michael T MadiganAbstract:Heliobacteria are a recently discovered group of anoxygenic phototrophic bacteria, first described in 1983. Heliobacteria contain bacteriochlorophyll g , a pigment unique to species of this group, and synthesize the simplest photosynthetic complexes of all known phototrophs. Also, unlike all other phototrophs, heliobacteria lack a mechanism for autotrophy and produce endospores. Four genera of heliobacteria containing a total of 10 species are known. Species of the genera Heliobacterium , Heliobacillus , and Heliophilum grow best at neutral pH, whereas species of Heliorestis are alkaliphilic. Heliobacterium , Heliobacillus , and Heliophilum species form one phylogenetic clade of heliobacteria, while Heliorestis species form a second within the phylum Firmicutes of the domain Bacteria . Heliobacteria have a unique ecology, being primarily terrestrial rather than aquatic phototrophs, and may have evolved a mutualistic relationship with plants, in particular, rice plants. The genome sequence of the thermophile Heliobacterium modesticaldum supports the hypothesis that heliobacteria are “minimalist phototrophs” and that they may have played a key role in the evolution of phototrophic bacteria.
-
Heliorestis convoluta sp. nov., a coiled, alkaliphilic Heliobacterium from the Wadi El Natroun, Egypt
Extremophiles, 2006Co-Authors: Marie Asao, Laurie A. Achenbach, Deborah O. Jung, Michael T MadiganAbstract:A morphologically distinct Heliobacterium, strain HH, was isolated from Lake El Hamra, a soda lake in the Wadi El Natroun region of northwest Egypt. Strain HH consisted of ring-shaped cells that remained attached after cell division to yield coils of various lengths. Strain HH showed several of the physiological properties of known heliobacteria and grouped in the Heliorestis clade by virtue of its phylogeny and alkaliphily. The closest relative of strain HH was the filamentous alkaliphilic Heliobacterium Heliorestis daurensis . However, genomic DNA:DNA hybridization results clearly indicated that strain HH was a distinct species of Heliorestis . Based on its unique phenotypic and genetic properties we describe strain HH here as a new species of the genus Heliorestis , H. convoluta sp. nov.
-
isolation and characterization of psychrophilic purple bacteria from antarctica
1999Co-Authors: Michael T MadiganAbstract:Several anoxygenic phototrophic bacteria (“anoxyphototrophs”) have been isolated and characterized from extreme environments. These include organisms from thermal [1], hypersaline [2-6], acidic [7,8], and alkaline [2,5,6] environments. In the author’s laboratory, thermophilic species of purple and green sulfur bacteria, Chromatium tepidum [9,10] and Chlorobium tepidum [11], respectively, and of heliobacteria, Heliobacterium modesticaldum [12], have been described; all of these organisms are capable of growth above 50°C. In addition, a variety of thermotolerant nonsulfur purple bacteria have been characterized, although none of these show growth above 50°C [13-15].
Shigeru Itoh - One of the best experts on this subject based on the ideXlab platform.
-
Excitonic Coupling on a Heliobacterial Symmetrical Type-I Reaction Center: Comparison with Photosystem I.
The journal of physical chemistry. B, 2020Co-Authors: Hirotaka Kitoh-nishioka, Yasuteru Shigeta, Shigeru Itoh, Akihiro KimuraAbstract:The excitonic couplings among 54 bacteriochlorophylls-g (BChl)-g, 4 BChl-g′, and 2 Chl-aF pigments were calculated in the type-I homodimeric reaction center (RC) of Heliobacterium modesticaldum (hR...
-
Theoretical Model of Exciton States and Ultrafast Energy Transfer in Heliobacterial Type I Homodimeric Reaction Center.
The journal of physical chemistry. B, 2018Co-Authors: Akihiro Kimura, Shigeru ItohAbstract:A simple theoretical model of exciton dynamics was proposed to interpret the fast excitation energy-transfer process in the type I homodimeric reaction center of Heliobacterium modesticaldum (hRC); this structure was recently identified and shown to resemble that of the plant/cyanobacterial photosystem I (PSI) reaction center. The exciton state model, which mainly relies on the geometries of 54 bacteriochlorophyll (BChl) g, 4 BChl-g′, and 2 chlorophyll (Chl) a on hRC and assumes constant site energy values for the pigments, reproduced the absorption spectrum of hRC rather well. The model also enabled numerical analysis of the exciton dynamics on hRC, which can be compared with the decay-associated spectra obtained by the laser spectroscopy experiments. The model indicates that the stronger transition–dipole moment on BChl-g contributes to the faster energy transfer due to the higher coherency of the delocalized exciton states on hRC compared to that on PSI that arranges Chl-a at almost homologous locations.
-
Light-Induced Electron Spin-Polarized (ESP) EPR Signal of the P800+ Menaquinone– Radical Pair State in Oriented Membranes of Heliobacterium modesticaldum: Role/Location of Menaquinone in the Homodimeric Type I Reaction Center
The journal of physical chemistry. B, 2018Co-Authors: Toru Kondo, Masami Kobayashi, Shigeru Itoh, Masahiro Matsuoka, Chihiro Azai, Hirozo Oh-okaAbstract:Function/location of menaquinone (MQ) was studied in the photosynthetic reaction center of Heliobacterium (Hbt.) modesticaldum (hRC), which is one of the most primitive homodimeric type I RCs. The spin-polarized electron paramagnetic resonance signals of light-induced radical pair species, which are made of oxidized electron donor bacteriochlorophyll g (P800+) and reduced menaquinone (MQ–) or iron–sulfur cluster (FX–), were measured in the oriented membranes of Hbt. modesticaldum at cryogenic temperature. The spectral shape of transient electron spin-polarized signal of P800+FX– radical pair state varied little with respect to the direction of the external magnetic field. It suggested a dominant contribution of the spin evolution on the precursor primary radical pair P800+A0– state with the larger isotropic magnetic exchange interaction J than the anisotropic dipole interaction D. The pure P800+MQ– signal was simulated by subtracting the effects of spin evolution during the electron-transfer process. It w...
-
Theoretical Model of Exciton States and Ultrafast Energy Transfer in Heliobacterial Type I Homodimeric Reaction Center
2018Co-Authors: Akihiro Kimura, Shigeru ItohAbstract:A simple theoretical model of exciton dynamics was proposed to interpret the fast excitation energy-transfer process in the type I homodimeric reaction center of Heliobacterium modesticaldum (hRC); this structure was recently identified and shown to resemble that of the plant/cyanobacterial photosystem I (PSI) reaction center. The exciton state model, which mainly relies on the geometries of 54 bacteriochlorophyll (BChl) g, 4 BChl-g′, and 2 chlorophyll (Chl) a on hRC and assumes constant site energy values for the pigments, reproduced the absorption spectrum of hRC rather well. The model also enabled numerical analysis of the exciton dynamics on hRC, which can be compared with the decay-associated spectra obtained by the laser spectroscopy experiments. The model indicates that the stronger transition–dipole moment on BChl-g contributes to the faster energy transfer due to the higher coherency of the delocalized exciton states on hRC compared to that on PSI that arranges Chl-a at almost homologous locations
-
Orientations of Iron–Sulfur Clusters FA and FB in the Homodimeric Type-I Photosynthetic Reaction Center of Heliobacterium modesticaldum
The journal of physical chemistry. B, 2016Co-Authors: Toru Kondo, Shigeru Itoh, Masahiro Matsuoka, Chihiro Azai, Hirozo Oh-okaAbstract:Orientations of the FA and FB iron–sulfur (FeS) clusters in a structure-unknown type-I homodimeric heriobacterial reaction center (hRC) were studied in oriented membranes of the thermophilic anaerobic photosynthetic bacterium Heliobacterium modesticaldum by electron paramagnetic resonance (EPR), and compared with those in heterodimeric photosystem I (PS I). The Rieske-type FeS center in the cytochrome b/c complex showed a well-oriented EPR signal. Illumination at 14 K induced an FB– signal with g-axes of gz = 2.066, gy = 1.937, and gx = 1.890, tilted at angles of 60°, 60°, and 45°, respectively, with respect to the membrane normal. Chemical reduction with dithionite produced an additional signal of FA–, which magnetically interacted with FB–, with gz = 2.046, gy = 1.942, and gx = 1.911 at 30°, 60°, and 90°, respectively. The angles and redox properties of FA– and FB– in hRC resemble those of FB– and FA–, respectively, in PS I. Therefore, FA and FB in hRC, named after their g-value similarities, seem to be...
Jan Amesz - One of the best experts on this subject based on the ideXlab platform.
-
endor and special triple resonance spectroscopy of photoaccumulated semiquinone electron acceptors in the reaction centers of green sulfur bacteria and heliobacteria
Biochemistry, 1999Co-Authors: Irine P Muhiuddin, Jan Amesz, Stephen E J Rigby, Michael C W Evans, Peter HeathcoteAbstract:Photoaccumulation at 205 K in the presence of dithionite produces EPR signals in anaerobically prepared membranes from Chlorobium limicola and Heliobacterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A1•-) photoaccumulated in photosystem I. We have used ENDOR and special TRIPLE resonance spectroscopy to demonstrate conclusively that these signals arise from menasemiquinone electron acceptors reduced by photoaccumulation. Hyperfine couplings to two protons H-bonded to the semiquinone oxygens have been identified by exchange of H. chlorum into D2O, and hyperfine couplings to the methyl group, and the methylene group of the phytyl side chain, of the semiquinone have also been assigned. The electronic structure of these menasemiquinones in these reaction centers is very similar to that of phyllosemiquinone in PSI, and shows a distorted electron spin density distribution relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the eff...
-
Spurious circular dichroism signals with intact cells of heliobacteria
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1994Co-Authors: Christof Francke, Stephan C.m. Otte, Jos C. Van Der Heiden, Jan AmeszAbstract:Abstract Strong anomalous circular dichroism (CD) signals were observed with suspensions of intact cells of the heliobacteria Heliobacillus mobilis and Heliobacterium chlorum. They were an order of magnitude larger than with isolated membranes at 6 K and changed dramatically with temperature. From the fact that suspensions of intact cells of heliobacteria showed linear dichroism (LD), it was concluded that the cells were oriented. The signals were proven to be artifacts, caused by coupling between LD and strain linear birefringence together with a smaller direct contribution of LD. The same effects were observed with oriented membrane fragments. The orientation of the cells was caused by gravity and could be eliminated by suspending the cells in an isopycnic solution. This way the real CD of intact cells could be measured. As expected, this spectrum was very similar to that of membrane fragments.
-
Energy transfer and trapping of excitations in membranes of Heliobacterium chlorum at 15 K
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1994Co-Authors: Paula I. Van Noort, T. J. Aartsma, Jan AmeszAbstract:Abstract The transfer of excitation energy in membrane fragments of Heliobacterium chlorum was studied at 15 K by picosecond transient absorption spectroscopy. Due to the high repetition rate of the laser pulses, the primary electron donor was in the oxidized state in essentially all reaction centers. The absorbance difference spectrum showed that excitations in the antenna rapidly accumulate on the long wavelength absorbing component bacteriochlorophyll (BChl) g 808. Analysis of the kinetics of excited antenna BChl g showed a lifetime of less than 1 ps for excitations on BChl g 778, whereas excited BChl g 793 showed a somewhat slower decay of about 2 ps. Time resolved anisotropy measurements showed a very rapid randomization of excitations amongst pigments with similar absorption spectrum within less than 1 ps. Kinetics as well as the absorbance difference spectrum showed that the decay of excitations accumulated on BChl g 808 was strongly inhomogeneous. About 50% of the excitations disappeared with a time constant of 4 ± 2 ps, while the others showed decays with time constants of 20 ± 5 ps and 100 ± 20 ps. The amplitude of the 20 ps component decreased with the energy density of the excitation pulses and this decay component is attributed, at least in part, to singlet-triplet quenching. The main relaxation process of excitations with a time constant of about 4 ps is most probably due to energy transfer to the oxidized reaction center. The 100 ps decay component is associated with BChls g 808 that are relatively isolated, either spatially or energetically.
-
Electron transfer in menaquinone-depleted membranes of Heliobacterium chlorum
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1993Co-Authors: F. A. M. Kleinherenbrink, Stephan C.m. Otte, Isamu Ikegami, A. Hiraishi, Jan AmeszAbstract:Abstract Treatment of membranes of Heliobacterium chlorum with diethyl ether at various levels of water saturation resulted in extraction of bacteriochlorophyll (BChl) g and menaquinones. Only a minor enrichment of P-798 with respect to antenna BChl g could be achieved, whereas menaquinone (vitamin K-2), the only quinone found in this species (Hiraishi, A. (1989) Arch. Microbiol. 151, 378–379), was essentially completely removed. Extraction of menaquinone, however, did not result in significant changes in electron transport. Electron transport to secondary electron acceptors was not impaired by wet ether extraction, either at room temperature or at low temperature. As in intact membranes, radical pair recombination and subsequent triplet formation were only observed under strongly reducing conditions. These results suggest that menaquinone is not an essential participant in the electron acceptor chain of heliobacteria.
-
Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures
Photochemistry and photobiology, 1993Co-Authors: F. A. M. Kleinherenbrink, Jan Amesz, P. Cheng, Robert E. BlankenshipAbstract:Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.
Robert E. Blankenship - One of the best experts on this subject based on the ideXlab platform.
-
Structural Analysis of Diheme Cytochrome c by Hydrogen− Deuterium Exchange Mass Spectrometry and Homology Modeling
2016Co-Authors: Ying Zhang, Hai Yue, Robert E. Blankenship, Erica L.-w. Majumder, Michael L. GrossAbstract:ABSTRACT: A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen−deuterium exchange mass spec-trometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cyto-chrome c (DHCC) protein from Heliobacterium modesticaldum is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homol-ogy modeling to yield a more functionally relevant structural model of DHCC. Cytochrome c’s are essential metalloproteins in theelectron-transfer chain of most living organisms, including all photosynthetic taxa. Metalloproteins move electrons around the cell to produce energetic compounds that drive cellular metabolism. Many of the photosynthetic electron-transfer pro-teins of interest belong to the cytochrome c family. Under
-
Structural Analysis of Diheme Cytochrome c by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling
2015Co-Authors: Ying Zhang, Hai Yue, Robert E. Blankenship, Erica L.-w. Majumder, Michael L. GrossAbstract:A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen–deuterium exchange mass spectrometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cytochrome c (DHCC) protein from Heliobacterium modesticaldum is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homology modeling to yield a more functionally relevant structural model of DHCC
-
Structural Analysis of Diheme Cytochrome c by Hydrogen–Deuterium Exchange Mass Spectrometry and Homology Modeling
Biochemistry, 2014Co-Authors: Ying Zhang, Erica L.-w. Majumder, Hai Yue, Robert E. Blankenship, Michael L. GrossAbstract:A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen–deuterium exchange mass spectrometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cytochrome c (DHCC) protein from Heliobacterium modesticaldum is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homology modeling to yield a more functionally relevant structural model of DHCC.
-
Structural analysis of diheme cytochrome c by hydrogen-deuterium exchange mass spectrometry and homology modeling.
Biochemistry, 2014Co-Authors: Ying Zhang, Erica L.-w. Majumder, Hai Yue, Robert E. Blankenship, Michael L. GrossAbstract:A lack of X-ray or nuclear magnetic resonance structures of proteins inhibits their further study and characterization, motivating the development of new ways of analyzing structural information without crystal structures. The combination of hydrogen–deuterium exchange mass spectrometry (HDX-MS) data in conjunction with homology modeling can provide improved structure and mechanistic predictions. Here a unique diheme cytochrome c (DHCC) protein from Heliobacterium modesticaldum is studied with both HDX and homology modeling to bring some definition of the structure of the protein and its role. Specifically, HDX data were used to guide the homology modeling to yield a more functionally relevant structural model of DHCC.
-
Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum.
Archives of biochemistry and biophysics, 2011Co-Authors: Hai Yue, Yisheng Kang, Hao Zhang, Xinliu Gao, Robert E. BlankenshipAbstract:Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.
Kevin E. Redding - One of the best experts on this subject based on the ideXlab platform.
-
Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum
Photosynthesis Research, 2019Co-Authors: Gregory S. Orf, Kevin E. ReddingAbstract:The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ∆pshA::cbp2p - aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. In addition, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This “ pshA rescue” strategy also provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.
-
Expression and characterization of cytochrome c _553 from Heliobacterium modesticaldum
Photosynthesis Research, 2014Co-Authors: Trevor S. Kashey, John B. Cowgill, Michael D. Mcconnell, Marco Flores, Kevin E. ReddingAbstract:Cytochrome c_553 of Heliobacterium modesticaldum is the donor to P_800 ^+, the primary electron donor of the heliobacterial reaction center (HbRC). It is a membrane-anchored 14-kDa cytochrome that accomplishes electron transfer from the cytochrome bc complex to the HbRC. The petJ gene encoding cyt c _553 was cloned and expressed in Escherichia coli with a hexahistidine tag replacing the lipid attachment site to create a soluble donor that could be made in a preparative scale. The recombinant cytochrome had spectral characteristics typical of a c -type cytochrome, including an asymmetric α-band, and a slightly red-shifted Soret band when reduced. The EPR spectrum of the oxidized protein was characteristic of a low-spin cytochrome. The midpoint potential of the recombinant cytochrome was +217 ± 10 mV. The interaction between soluble recombinant cytochrome c _553 and the HbRC was also studied. Re-reduction of photooxidized P_800 ^+ was accelerated by addition of reduced cytochrome c _553. The kinetics were characteristic of a bimolecular reaction with a second order rate of 1.53 × 10^4 M^−1 s^−1 at room temperature. The rate manifested a steep temperature dependence, with a calculated activation energy of 91 kJ mol^−1, similar to that of the native protein in Heliobacillus gestii cells. These data demonstrate that the recombinant soluble cytochrome is comparable to the native protein, and likely lacks a discrete electrostatic binding site on the HbRC.
-
Temporal and spectral characterization of the photosynthetic reaction center from Heliobacterium modesticaldum
Photosynthesis Research, 2013Co-Authors: Adrien Chauvet, Steven Romberger, John H Golbeck, Josephine Sarrou, Su Lin, Sergei Savikhin, Kevin E. ReddingAbstract:A time-resolved spectroscopic study of the isolated photosynthetic reaction center (RC) from Heliobacterium modesticaldum reveals that thermal equilibration of light excitation among the antenna pigments followed by trapping of excitation and the formation of the charge-separated state P_800 ^+A_0 ^– occurs within ~25 ps. This time scale is similar to that reported for plant and cyanobacterial photosystem I (PS I) complexes. Subsequent electron transfer from the primary electron acceptor A_0 occurs with a lifetime of ~600 ps, suggesting that the RC of H. modesticaldum is functionally similar to that of Heliobacillus mobilis and Heliobacterium chlorum . The (A_0 ^– − A_0) and (P_800 ^+ − P_800) absorption difference spectra imply that an 8^1-OH-Chl a _ F molecule serves as the primary electron acceptor and occupies the position analogous to ec3 (A_0) in PS I, while a monomeric BChl g pigment occupies the position analogous to ec2 (accessory Chl). The presence of an intense photobleaching band at 790 nm in the (A_0 ^– − A_0) spectrum suggests that the excitonic coupling between the monomeric accessory BChl g and the 8^1-OH-Chl a _ F in the heliobacterial RC is significantly stronger than the excitonic coupling between the equivalent pigments in PS I.
-
Purification of the photosynthetic reaction center from Heliobacterium modesticaldum
Photosynthesis Research, 2012Co-Authors: Iosifina Sarrou, John Cowgill, Daniel Brune, Steven Romberger, John H Golbeck, Zahid Khan, Kevin E. ReddingAbstract:We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum . HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006 ; Romberger et al., Photosynth Res 104:293–303, 2010 ) are now identified by mass spectrometry to be the membrane-bound cytochrome c _553 and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g , two BChl g ′, two 8^1-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P_800 ^+F _X ^− state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s^−1 under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.