Podcasts about Rhodobacter

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547267v1?rss=1 Authors: Zamal, M. Y., Venkataramana, C., Subramanyam, R. Abstract: In the phototrophic alphaproteobacteria, photosynthesis is performed by pigment-protein complexes, including the light-harvesting complexes known as LH1 and LH2. The photosystem also encompasses carotenoids to assist in well-functioning of photosynthesis. Most photosynthetic bacteria are exposed to various abiotic stresses, and here, the Rhodobacter (R.) alkalitolerans were extracted from the alkaline pond. We report the comparative study of photosynthetic apparatus of R. alkalitolerans in various light intensities in relation to this bacterium's high pH tolerance ability. We found that as the light intensity increased, the stability of photosystem complexes decreased in normal pH (npH pH 6.8{+/-}0.05) conditions, whereas in high pH (hpH pH 8.6{+/-}0.05) acclimation was observed. The content of bacteriochlorophyll a, absorbance spectra, and circular dichroism data shows that the integrity of photosystem complexes is less affected in hpH compared to npH conditions. Sucrose density and LP-BN of photosystem complexes also shows that LH2 is more affected in npH than hpH, whereas RC-LH1 monomer or dimer has shown interplay between monomer and dimer in hpH although the dimer and monomer both increased in npH. Additionally, the phosphatidylcholine (PC) levels have increased in hpH conditions. Moreover, qPCR data showed that the subunit -c of ATPase levels was overexpressed in hpH. Consequently, the P515 measurement shows that more ATP production is required in hpH, which dissipates the protons from the chromatophore lumen. This could be the reason the photosystem protein complex destabilized due to more lumen acidification. To maintain homeostasis in hpH, the antiporter NhaD expressed more than in the npH condition. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

How does an engineer approach microbial genetics? cworks with microbes of all kinds to optimize metabolic and agricultural systems. Here he discusses his work with Rhodobacter to make biofuels and for membrane protein expression, with Agrobacterium and plant pathogenic viruses to make drought-resistant plants, and with Clostridium and yeast cocultures for lignocellulose digestion. Take the listener survey at asm.org/mtmpoll Full shownotes at asm.org/mtm Links for this Episode: Wayne Curtis Lab site at Penn State University PLoS One: Molecular Cloning, Overexpression, and Characerization of a Novel Water Channel protein from Rhodobacter sphaeroides Protein Expression and Purification: Advancing Rhodobacter sphaeroides as a Platform for Expression of Functional Membrane Proteins Biotechnology for Biofuels: Consortia-Mediated Bioprocessing of Cellulose to Ethanol with a symbiotic Clostridium phytofermentans/Yeast Co-Culture HOM Tidbit: Genentech “Cloning Insulin” blog HOM Tidbit: Genentech press release announcing insulin cloning  

This episode: Phages may be passing through the barriers in our body all the time! Thanks to Dr. Jeremy Barr for his contribution! Download Episode (13.2 MB, 14.5 minutes) Show notes: Microbe of the episode: Rhodobacter virus RcCronus Journal Paper: Nguyen S, Baker K, Padman BS, Patwa R, Dunstan RA, Weston TA, Schlosser K, Bailey B, Lithgow T, Lazarou M, Luque A, Rohwer F, Blumberg RS, Barr JJ. 2017. Bacteriophage Transcytosis Provides a Mechanism To Cross Epithelial Cell Layers. mBio 8:e01874-17. Other interesting stories: Yeast species uses same codon for two different amino acids (paper)   Email questions or comments to bacteriofiles at gmail dot com. Thanks for listening! Subscribe: Apple Podcasts, RSS, Google Play. Support the show at Patreon, or check out the show at Twitter or Facebook

Mon, 28 Jul 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18573/ https://edoc.ub.uni-muenchen.de/18573/1/Dominguez_Pablo_Nahuel.pdf Dominguez, Pablo Nahuel ddc:530, ddc:500, Fakultät für Physi

The natural design of the photosystems of plants and photosynthetic bacteria using chlorophylls (Chls) or bacteriochlorophylls (BChls) as photoreceptors are robust. The basic principles of the biological system of light-harvesting complex 2 (LH2) are studied with the use of natural and model sequences expressed in vivo in modified Rhodobacter (Rb) sphaeroides strains. Three aspects have been explored in the thesis: (1) BChl’s macrocycle-protein interactions, (2) BChl’s phytol-protein interactions underlying the structural and functional assembly of the pigment-protein complexes, and (3) LH2-lipid interactions and the role of these interactions in photosynthetic membrane morphogenesis. BChls’ macrocycle-protein interactions: Residues at the immediate BChl-B850/protein interface are found to have little effect on specifying the BChl-B850 array, and their light-harvesting activity in LH2. Nevertheless, these residues are important for the structural thermal stability. With the use of ‘rescue’ mutagenesis of the model BChl binding site, the hydrogen-bond between αSer -4 and the C131 keto carbonyl group of βBChl-B850 is shown to be a crucial motif for driving the assembly of model LH2 complex. Possibilities for residue modifications are limited in the β-subunits as compared to the α-subunits, which suggests that the two polypeptides have distinct roles in complex assembly. In the β-subunits, there are residues detected adjacent to the BChl-B850 site which are critical for the assembly of LH2. BChls’ phytol-protein interactions: Mutagenesis of residues closely interacting with the BChl-B850 phytol moiety result in the pronounced loss of BChl-B800 from LH2. Dephytylation of bound BChls within assembled LH2 to BChlides also resulted in the loss of BChl-B800 and destabilisation of LH2 structural assembly. Thus, the phytol chains were shown to be important for optimal pigment binding, especially for BChl-B800; which appears to be highly sensitive to the proper packing of the phytols. The pattern of phytol interactions with their surrounding environments are significantly different for α- and β-ligated (B)Chls. The phytols of β-ligated (B)Chls, as opposed to α-ligated (B)Chls, have ample and specific interactions with residues of the binding helix which may contribute to the tertiary interactions of helices. LH2-lipids interactions: Phospholipid determination of LH2 only expressing strains of Rb sphaeroides shows that the nonbilayer-forming phospholipid, phosphatidylethanolamine (PE) is present in elevated amounts in the intracytoplasmic membranes and in the immediate vicinity of the LH2 complex. In combination with βGlu -20 residue and the carotenoid headgroup at the N-terminus of the transmembrane β-helices is shown to influence the composition of lipids surrounding LH2. Specific local interactions between LH2 protein and lipids not only promote LH2 protein stability but appear to modulate the morphology of intracytoplasmic membranes. Based on these findings, the presence of LH2-lipid specificity is postulated. The approach of using model αβ-sequences with simplified pigment binding sites allows us to study the underlying factors involved in LH2 assembly and function. This gives rise to a better understanding of the interplay between BChl, apoproteins and membrane lipids in the assembly of a highly efficient light-harvesting complex in its native lipid-environment.

The presented thesis has focused on the interactions between protein and pigments in photosynthetic membrane proteins, and the significance of these interactions in membrane protein assembly. The thesis has been divided into 3 Chapters, two are focused on the interactions between (bacterio)chlorophyll and proteins, and one is focused on the interactions, between carotenoid and proteins. In order to explore these interactions model proteins have been designed based on the peripheral antenna of Rhodobacter sphaeroides. In the model LH2 complexes, portions of the transmembrane helices, in particular, at the pigment binding sites, are replaced simplified alternating by alanine-leucine stretches. In the model sequence context, the effects of particular amino acids are amplified, and thus allow for convenient identification of potentially critical interaction motifs. This approach is employed to study the factors that contribute to pigment binding and pigment-protein assembly. To confirm the significance of thus identified motifs, they are subsequently also examined in the WT sequence context. In Chapter 3, it is shown that the residue at position -4 of the beta-subunit has a critical structural role for the proper organisation of the excitonically coupled BChl dimer in the antenna complex. In WT LH2, the residue at this position makes an H-bond to the C131 keto carbonyl group of one of the dimeric BChl molecules. The potential importance of such a H-bonding motif at the BChl/protein interface is demonstrated by use of the model LH2 in which the H-bond drives the folding and assembly of this transmembrane BChl-protein. The structural role of this residue at the BChl/protein interface is further demonstrated by the linear correlation between the LH2 spectral tuning and the residue-BChl contact. In Chapter 4, the aspect of diastereotopic ligation to the central Mg of BChl is explored, in particular, the consequences of BChl-ligation for folding and assembly of BChl-proteins. The analysis of H-bonding patterns in Chl-binding photosystem I and II showed that H-bonding at the (B)Chl-protein interface is structurally distinct depending on the ligation type. In essence, the C131 keto groups of (B)Chl ligated in the beta-position, contrary to those ligated in the betaposition, are frequently employed to associate Chl-helix units and thus involved in tertiary interactions. Disruption of such H-bonding interactions by site directed mutagenesis significantly altered the structural stability and assembly of the LH2 complex in the membrane. These findings suggest that H-bonding to -ligated bacteriochlorophyll is a key structural motif for the correct assembly of (bacterio)chlorophyll proteins. In Chapter 5, it is shown by mutational analysis of the carotenoid binding pocket of native and model LH2 complexes that the aromatic residues, in particular phenylalanine, are a key factor for carotenoid binding. The phenylalanine not only contributes to the stable Car binding but also lock the Car into a particular molecular configuration. The importance of aromatic residues in Car binding is further supported by statistical analyses of the plant photosystems which show that phenylalanine residues are frequently in the close vicinity of Car moelcules. This study provides, to the best of our knowledge, the first experimental evidence for the central role of aromatic residues in carotenoid binding and functional specification.

Ziel dieser Arbeit war zum Einen die Synthese von neuartigen (Bakterio-)Chlorophyll-Derivaten zur Einlagerung in Proteine und ihre Charakterisierung in Lösung, zum Anderen Bindungsstudien an Komplexen dieser Derivate mit modularen Proteinen und dem Lichtsammler-Komplex 1 aus Rhodobacter sphaeroides. 1.) Darstellung von Fe-(Bakterio-)Pheophytinen: Es wurde ein Verfahren etabliert, das die Metallierung von Pheophytin a, Bakteriopheophytin a und deren Derivate mit Eisen ermöglicht. Zusätzlich wurde diese Methode so weit modifiziert und optimiert, dass ausgehend vom Mohrschen Salz auch die Einlagerung von 57Fe möglich ist, wodurch eine Erweiterung der spektroskopischen Methoden (Mößbauer-Spektroskopie) erreicht wird. Da die Fe Komplexe nicht mit den für (Bakterio-)Chlorophyll-Derivate etablierten Methoden gereinigt werden können, wurde für diese Komplexe ein neues Chromatographiesystem entwickelt. 2) Spektroskopische Untersuchung der Fe-(Bakterio-)Pheophytine: Es ist bekannt, dass Fe-Porphyrine leicht zu µ-oxo-Komplexen (Fe(III)(B)Phe a)2O dimerisieren und das Zentralion in zwei Oxidationsstufen (+2 und +3) vorliegen kann. Die Dimerisierung des Fe Phe und Fe-BPhe wurde durch Säure-Base-Titration absorptionsspektroskopisch untersucht. In aerober Lösung liegt das Zentralmetall des Fe-(B)Phe dreiwertig vor ((Fe(III)(B)Phe a)Cl). Dieses lässt sich „klassisch“ mit Na-Dithionit allein durch Ligandierung mit Pyridin zum zweiwertigen Fe(II)(B)Phe a reduzieren. Die drei Zustände der Fe-Komplexe (Fe(III)(B)Phe a)Cl, (Fe(III)(B)Phe a)2O und Fe(II)(B)Phe a wurden durch ESR- und Absorptionsspektroskopie charakterisiert. Die Oxidationsstufen der drei Zustände wurden für 57Fe-Me-Pheid a durch Mößbauerspektroskopie bestätigt. 3) Einlagerung von Fe-Bakteriopheophytin a in LH1 von Rhodobacter sphaeroides: Zur Untersuchung, ob Fe-BPhe von BChl-Bindungstaschen akzeptiert wird, wurde versucht Fe-BPhe ins LH1 von Rb. sphaeroides einzulagern. Die Ergebnisse dieser Untersuchungen deuten zwar auf einen Einbau von Fe-BPhe hin, allerdings nur in sehr geringem Maß. Zur Quantifizierung des Fe-BPhe-Gehalts wurden verschiedene Methoden getestet. Der einfachste und vielversprechendste Weg war die Differenzabsorptionsspektroskopie, bei der die unterschiedliche Absorption von µ-oxo-Dimer und Monomer des Fe-BPhe ausgenützt wird. 4) Darstellung von Formyl-(Bakterio-)Chlorophyll-Derivaten: Eine kovalente Bindung von Chlorophyll-Derivaten an synthetische Peptide ist durch die Kopplung von Formyl-Gruppen mit einem modifizierten Lysin-Rest unter Bildung eines Oxims möglich. [3 Formyl]-Me-Pheid a konnte durch oxidative Spaltung des C-3-Vinyl des Chlorophyll a mit Ozon hergestellt werden. Ebenfalls mit Ozon konnte die Phytyl-Doppelbindung von Pheophytin a unter Bildung des Ethanal-Pheid a erreicht werden. Somit stehen insgesamt drei Chlorophyll-Derivate für die kovalente Bindung an synthetische Peptide zur Verfügung, welche die Formyl-Gruppen an verschiedenen Positionen tragen, wodurch eine unterschiedliche Orientierung der Pigmente im Protein erreicht werden kann. Es wurde versucht, in Analogie zum Pheophytin a das [3-Vinyl]-Me-BPheid a mit Ozon zu spalten. In Folge der leichten Oxidation des Makrozyklus lieferte diese Reaktion das Zielprodukt [3-Formyl]-Me-BPheid a nur in äußerst geringen Mengen, so dass diese Methode für die präparative Synthese dieser Verbindung nicht geeignet ist. 5) Nicht-kovalente Bindung von [M]-BPheid (M = Ni, Zn, Fe) in synthetische modulare Proteine (MOP): Ni-, Zn-, und Fe-BPhe wurden auf ihre Komplexbildung mit 216 verschiedenen, synthetischen Vier-Helix-Bündel-Proteinen untersucht. Die [M] BPheid-MOP-Komplexe wurden absorptionsspektroskopisch auf die Stärke der Bindung, die Koordination des Zentralmetalls und auf die Hydrophobizität der Umgebung untersucht. Alle MOP binden [M] BPheid in sehr unterschiedlichem Maße. Stärke und Art der Bindung werden in erster Linie durch die Bindehelix bestimmt. Eine quantitative Modulation findet allerdings auch durch die Abschirmhelix statt. Ni-BPheid zeigt in den Komplexen drei mögliche Koordinationszustände (nc = 4, 5, 6). Eine hohe Koordinationszahl geht immer mit einer stabilen Bindung und einer schmalen Qy Bande einher. Zn-BPheid ist in allen Komplexen fünffach koordiniert, das Fe-BPheid vierfach. Qualitativ zeigen alle drei Pigmente ein gleiches Muster in Bezug auf das Bindungsverhalten, so dass ausgehend von Häm-Bindungstaschen die Bildung von BChl-Bindungstaschen bestätigt werden konnte.

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P* becomes 4–5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll BA with its environment appears to be altered, possibly because of a change in its position.

Zero field absorption detected magnetic resonance hole burning measurements were performed on photosynthetic reaction centers of the bacteria Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis. Extrapolation to zero microwave power yielded pseudohomogeneous linewidths of 2.0 MHz for Rhodopseudomonas viridis, 1.0 and 0.9 MHz for the protonated forms of Rhodobacter sphaeroides R26 with and without monomer bacteriochlorophyll exchanged, and 0.25 MHz as an upper limit for fully deuterated reaction centers of Rhodobacter sphaeroides R26. The measured linewidths were interpreted as being due to unresolved hyperfine interaction between the nuclear spins and the triplet electron spin, the line shape being determined by spectral diffusion among the nuclei. The difference in linewidths between Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis is then explained by triplet delocalization on the special pair in the former, and localization on one dimer half on the latter. In the fully deuterated sample, four quadrupole satellites were observed in the hole spectra arising from the eight 14N nitrogens in the special pair. The quadrupole parameters seem to be very similar for all nitrogens and were determined to =1.25±0.1 MHz and =0.9±0.1 MHz. The Journal of Chemical Physics is copyrighted by The American Institute of Physics.

Sat, 1 Jan 1994 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3781/1/92.pdf Oesterhelt, Dieter; Zinth, Wolfgang; Lauterwasser, Christoph; Holzapfel, Wolfgang; Finkele, Ulrich; Stilz, Hans Ulrich

15N-substituted bacteriochlorophyll a (BChl a) was extracted from the cells of Rhodobacter sphaeroides 2.4.1 grown in a medium containing 15N-ammonium sulfate and yeast concentrate. The T1 Raman spectra of 14N-and 15N-BChl a were obtained as the difference spectra of high-power minus low-power of one-color, pump-and-probe measurements using 420 nm, 5 ns pulses. A set of empirical assignments of the T1 Raman lines was made, based on shifts upon 14N→15N substitution. The S0 Raman spectra of the two BChls were also obtained by using the 457.9 nm cw beam, and a set of assignments of the S0 Raman lines was given for comparison.

A series of substituted bacteriochlorophyll molecules, all used in reconstitution experiments of reaction centers of Rhodobacter sphaeroides (Struck et al. Biochim. Biophys. Acta 1991, 1060, 262-270), were characterized by EPR, electron-nuclear double (ENDOR), and electron-nuclear-nuclear triple (TRIPLE) resonance spectroscopy in their monomeric radical cation states. Effects of different substituents at position 3 in the porphyrin macrocycle were considered, especially for two «crosslinks» between plant and bacterial chlorophylls. These are 3-vinylbacteriochlorophyll where the «bacteria» acetyl group at position 3 was substituted by vinyl and 3-acetylchlorophyll where the «plant» vinyl group was substituted by acetyl

The modification of reaction centers from Rhodobacter sphaeroides by the introduction of pheophytins instead of bacteriopheophytins leads to interesting changes in the primary photosynthetic reaction: long-living populations of the excited electronic state of the special pair P* and the bacteriochlorophyll anion B−A show up. The data allow the determination of the energetics in the reaction center. The free energy of the first intermediate P+B−A, where the electron has reached the accessory bacteriochlorophyll BA lies ≈ 450 cm−1 below the initially excited special pair P*.

The primary photosynthetic reactions in whole membranes of the antenna-deficient mutant strain U43 (pTXA6–10) of Rhodobacter capsulatus are studied by transient absorption and emission spectroscopy with subpicosecond time resolution. Extensive similarities between the transient absorption data on whole membranes and on isolated reaction centers support the idea that the primary processes in isolated reaction centers are not modified by the isolation procedure.

Triplet state electron paramagnetic resonance (EPR) experiments have been carried out at X-band on Rb. sphaeroides R-26 reaction centers that have been reconstituted with the carotenoid, spheroidene, and exchanged with 132-OH-Zn-bacteriochlorophyll a and [3-vinyl]-132-OH-bacteriochlorophyll a at the monomeric, lsquoaccessoryrsquo bacteriochlorophyll sites BA,B or with pheophytin a at the bacteriopheophytin sites HA,B. The primary donor and carotenoid triplet state EPR signals in the temperature range 95–150 K are compared and contrasted with those from native Rb. sphaeroides wild type and Rb. sphaeroides R-26 reaction centers reconstituted with spheroidene. The temperature dependencies of the EPR signals are strikingly different for the various samples. The data prove that triplet energy transfer from the primary donor to the carotenoid is mediated by the monomeric, BChlB molecule. Furthermore, the data show that triplet energy transfer from the primary donor to the carotenoid is an activated process, the efficiency of which correlates with the estimated triplet state energies of the modified pigments.

The primary electron transfer in reaction centers of Rhodobacter sphaeroides is studied by subpicosecond absorption spectroscopy with polarized light in the spectral range of 920-1040 nm. Here the bacteriochlorophyll anion radical has an absorption band while the other pigments of the reaction center have vanishing ground-state absorption. The transient absorption data exhibit a pronounced 0.9-ps kinetic component which shows a strong dichroism. Evaluation of the data yields an angle between the transition moments of the special pair and the species related with the 0.9-ps kinetic component of 26 +/- 8 degrees. This angle compares favorably with the value of 29 degrees expected for the reduced accessory bacteriochlorophyll. Extensive transient absorbance data are fully consistent with a stepwise electron transfer via the accessory bacteriochlorophyll.

Nonlinear polarization spectroscopy in the frequency domain allows rate constant determinations of fast electronic energy and phase relaxations together with characterization of the type of line broadening. Application of this method to the B850 component of the isolated B800–850antenna ofRhodobacter sphaeroides at room temperature shows that B850 is inhomogeneously broadened, with homogeneous widths between 30 and 200 cm−1, depending on the spectral position of the subforms. The corresponding phase relaxation times are clearly in the subpicosecond range. There is also indication of an up-to-now unspecified1–5 ps energy relaxation channel per subunit.

The spontaneous emission of reaction centers from native and mutated Rhodobacter sphaeroides and from wild type Chloroflexus aurantiacus is investigated by fluorescence up-conversion with high temporal resolution. The time constant of 0.9 ps previously observed in transient absorption experiments on wild type reaction centers of Rhodobacter sphaeroides does not appear in the emission experiment. However, all investigated reaction centers display a biexponential decay of the emission with time constants in the 2 ps to 25 ps range. The experimental results are discussed within the frame of different reaction models including a possible sample heterogeneity or a transient electron transfer to the inactive pigment branch.

The spontaneous emission of reaction centers from native and mutated Rhodobacter sphaeroides and from wild type Chloroflexus aurantiacus is investigated by fluorescence up-conversion with high temporal resolution. The time constant of 0.9 ps previously observed in transient absorption experiments on wild type reaction centers of Rhodobacter sphaeroides does not appear in the emission experiment. However, all investigated reaction centers display a biexponential decay of the emission with time constants in the 2 ps to 25 ps range. The experimental results are discussed within the frame of different reaction models including a possible sample heterogeneity or a transient electron transfer to the inactive pigment branch.

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g. Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides,RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+QA− or P+QB−, respectively; (b) the kinetics of electron transfer from QA− to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (τ = 3.5 ps), followed by reduction of the bacteriopheophytin (τ = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (τ = 410 ± 30 and 47 ± 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type). EPR studies of the isolated RCs showed a characteristic g = 1.82 signal for the QA semiquinone coupled to the iron atom, which was indistinguishable from the wild type. It is concluded that GluM234 is not essential to the normal functioning of the acceptor quinone complex in bacterial RCs and that the role of bicarbonate in PS II is distinct from the role of this residue in bacterial RCs.

The primary electron transfer has been investigated by femtosecond time-resolved absorption spectroscopy in two chemically modified reaction centers (RC) of Rhodobacter sphaeroides, in which the monomeric bacteriochlorophylls BA and BB have both been exchanged by 13(2)-hydroxybacteriochlorophyll a or [3-vinyl]-13(2)-hydroxybacteriochlorophyll a. The kinetics of the primary electron transfer are not influenced by the 13(2)-hydroxy modification. In RCs containing [3-vinyl]-13(2)-hydroxybacteriochlorophyll a the primary rate is reduced by a factor of 10.

The primary electron transfer (ET) in reaction centers (RC) of Rhodobacter sphaeroides is investigated as a function of temperature with femtosecond time resolution. For temperatures from 300 to 25 K the ET to the bacteriopheophytin is characterized by a biphasic time dependence. The two time constants of τ1=3.5±0.4 ps and τ2=1.2±0.3 ps at T=300 K decrease continously with temperature to values of τ1=1.4±0.3 ps and τ2=0.3±0.15 ps at 25 K. The experimental results indicate that the ET is not thermally activated and that the same ET mechanisms are active at room and low temperatures. All observations are readily rationalized by a two-step ET model with the monomeric bacteriochlorophyll as a real electron carrier.

Pigments of borohydride-treated reaction centers of Rhodobacter sphaeroides R 26 and Rhodopseudomonas viridis were analyzed by HPLC with polychromatic detection. In both species, pigment composition and contents were unchanged. Reaction centers from Rhodobacter sphaeroides R26 were prepared in which bacteriochlorophylls (BA,B) and bacteriopheophytins (HA,B) were exchanged with their potential borohydride products reduced at C-31. [3-Hydroxyethyl]-BChl a exchanges selectively into the BA,B pockets, and 31-OH-BPh a to the HA,B pockets. Stable reaction centers are obtained in both cases. A comparison of the absorption and circular dichroism spectra of reaction centers after exchange with 31-OH pigments, and of borohydride-modified reaction centers, reveal distinct differences. It is concluded that during borohydride reduction none of the pigments is chemically modified or extracted from the reaction centers.

Femtosecond spectroscopy was used in combination with site-directed mutagenesis to study the influence of tyrosine M210 (YM210) on the primary electron transfer in the reaction center of Rhodobacter sphaeroides. The exchange of YM210 to phenylalanine caused the time constant of primary electron transfer to increase from 3.5 f 0.4 ps to 16 f 6 ps while the exchange to leucine increased the time constant even more to 22 f 8 ps. The results suggest that tyrosine M210 is important for the fast rate of the primary electron transfer.

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B. State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.

Monomeric bacteriochlorophylls BA and BB in photosynthetic reaction centers from Rhodobacter sphaeroides R26 were exchanged with (132-hydroxy-) bacteriochlorophylls containing a 3-vinyl- or 3-(α-hydroxyethyl)-substituent instead of the 3-acetyl group. The corresponding binding sites must be tolerant to the introduction of the polar residue at C-132 and modifications of the 3-acetyl group. According to HPLC analysis, the exchange with both pigments amounts to less-than over equal to 50% of the total BChl contained in the complex, corresponding to less-than over equal to 100% of the monomeric BChl aBA,B. The absorption spectra show significant changes in the Qx and Qy-region of the monomeric bacteriochlorophylls. By contrast, the absorption of the primary donor (P870) and reversible photobleaching is retained. The circular dichroism is also unchanged in the 870 nm region. The positive cd band located at around 800 nm in native reaction centers, shifts with the (blue-shifted) Qy absorption(s) of BA and/or BB, whereas the position of the negative one remains nearly unaffected. The data indicate that the latter is the upper excitonic band of the primary donor, and that there is little interaction of the monomeric BA/BB with the primary donor.

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond time-resolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 +/- 0.4 ps to the accessory bacteriochlorophyll B. State P+B- decays with a time constant of 0.9 +/- 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H- to the quinone QA within 220 +/- 40 ps.

Mon, 1 Jan 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3758/1/3758.pdf Zinth, Wolfgang; Stilz, Hans Ulrich; Scheer, Hugo; Oesterhelt, Dieter; Michel, H.; Kaiser, Wolfgang; Buchanan, S.; Holzapfel, Wolfgang; Hamm, P.; Lauterwasser, Christoph; Finkele, Ulrich; Dressler, K.

Mon, 1 Jan 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3755/1/3755.pdf Oesterhelt, Dieter; Zinth, Wolfgang; Lauterwasser, Christoph; Holzapfel, Wolfgang; Finkele, Ulrich; Stilz, Hans Ulrich

Incubation of photosynthetic reaction centers from Rhodobacter sphaeroides R26 with exogenous 132-OH-bacteriochlorophyll ap or aGG according to Scheer et al. (1987) results in the exchange of endogenous bacteriochlorophyll ap. The exchange amounts to less-than-or-equals, slant 50% according to HPLC analysis, corresponding to a complete replacement of the ‘monomeric’ bacteriochlorophylls, bm and bl, by exogenous pigment. The absorption spectra show small, but distinct changes in the Qx-region of the bacteriochlorophylls, and bleaching of the modified reaction centers is retained. The corresponding binding sites must be accessible from the exterior, and allow for the introduction of a polar residue at C-132. This is supported by the observation of side reactions of the endogenous ‘monomeric’ bacteriochlorophylls within the reaction center pigments, e.g. epimerization and hydroxylation at C-132.

Mon, 1 Jan 1990 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3759/ http://epub.ub.uni-muenchen.de/3759/1/3759.pdf Gray, Kevin A.; Farchaus, J. W.; Wachtveitl, J.; Breton, J.; Finkele, Ulrich; Lauterwasser, Christoph; Zinth, Wolfgang; Oesterhelt, Dieter Gray, Kevin A.; Farchaus, J. W.; Wachtveitl, J.; Breton, J.; Finkele, Ulrich; Lauterwasser, Christoph; Zinth, Wolfgang und Oesterhelt, Dieter (1990): The role of tyrosine M210 in the initial charge separation in the reaction center of Rhodobacter sphaeroides. In: Michel-Beyerle, M.E. (Hrsg.), Springer Series in Biophysics: Reaction Centers of Photosynthetic Bacteria. Bd. 6, Springer: Berlin, pp. 251-264.

The primary light-induced charge separation in reaction centers of Rhodobacter sphaeroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 nm could only be explained by a kinetic model which uses three time constants. This finding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone.

Sun, 1 Jan 1989 12:00:00 +0100 https://epub.ub.uni-muenchen.de/3575/1/3575.pdf Finkele, Ulrich; Holzapfel, Wolfgang; Zinth, Wolfgang

The primary light-induced charge separation in reaction centers of Rhodobacter sphaeroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 nm could only be explained by a kinetic model which uses three time constants. This finding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone.

Reaction centers from Rhodobacter sphaeroides have been modified by treatment with sodium borohydride similar to the original procedure [Ditson et al., Biochim. Biophys. Acta 766, 623 (1984)], and investigated spectroscopically and by gel electrophoresis. (1) Low temperature (1.2 K) absorption, fluorescence, absorption- and fluorescence-detected ODMR, and microwave-induced singlet-triplet absorption difference spectra (MIA) suggest that the treatment produces a spectroscopically homogeneous preparation with one of the ‘additional’ bacteriochlorophylls being removed. The modification does not alter the zero field splitting parameters of the primary donor triplet (TP870). (2) From the circular dichroism and Raman resonance spectra in the1500–1800 cm-1 region, the removed pigment is assigned to BchlM, e.g. the "extra" Bchl on the "inactive" M-branch. (3) A strong coupling among all pigment molecules is deduced from the circular dichroism spectra, because pronounced band-shifts and/or intensity changes occur in the spectral components assigned to all pigments. This is supported by distinct differences among the MIA spectra of untreated and modified reaction centers, as well as by Raman resonance. (4) The modification is accompanied by partial proteolytic cleavage of the M-subunit. The preparation is thus spectroscopically homogeneous, but biochemically heterogenous.