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W Nitschke

Publications and source records attributed to W Nitschke.

At least 37 records · Page 2Linked to original sources
PubMed1997

Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553.

Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.

Chlorobi↗
PubMed1996

Membrane-bound c-type cytochromes in Heliobacillus mobilis. Characterisation by EPR and optical spectroscopy in membranes and detergent-solubilised material.

The spectral and electrochemical parameters, as well as the orientations of the heme plane with respect to the membrane plane, of the c-type hemes present in membrane fragments from Heliobacillus mobilis were characterised by optical and EPR spectroscopy. Cytochrome C53, was thereby shown to represent at least four and possibly five heme species with the following characteristics: Em = -60 mV +/- 10 mV, g, = 2.92, 60 degrees; Em = +90 mV +/- 10 mV, g, = 2.92, 90 degrees; Em = +120 mV +/- 20 mV, g, = 3.03; and Em = +170 mV +/- 20 mV, g, = 3.03. The latter component may correspond to two hemes with redox midpoint potentials of Em = +160 mV +/- 20 mV and Em = +180 mV +/- 20 mV (all Em values at pH 7.0). For the heme species having g, peaks at g approximately 3.03, determination of individual orientations was precluded due to the superposition of several differently oriented hemes. About one copy of each heme was found to be present per photosynthetic reaction centre, with the exception of the +120 mV component for which a stoichiometry of 2 hemes/reaction centre was obtained. The heme proteins were detergent-solubilised and partially purified. Three c-type cytochromes that migrated with apparent molecular masses of 18, 29 and 50 kDa were detected on SDS/PAGE. Optical redox titrations at pH 7.0 showed redox midpoint potentials of +160 mV +/- 10 mV for the 18-kDa cytochrome, and -60 mV +/- 10 mV, with possible contributions around +160 mV, for the 50-kDa cytochrome. A tentative attribution of heme species observed in membranes to the isolated heme proteins is presented. The results obtained on H. mobilis are compared with those reported for green sulphur bacteria.

Bacteria↗
PubMed1996

Characterisation of a soluble cytochrome c4 isolated from Thiobacillus ferrooxidans.

A soluble c-type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an alpha-peak wavelength of 552 nm, a molecular mass of 21 193 Da (as determined by mass spectroscopy), and a pI value of 9. N-terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em = 385 mV +/- 20 mV and Em = 480 mV +/- 20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz = 3.1 and gz = 3.35. The gz = 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half-times. All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c-type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.

Amino Acid Sequence↗
PubMed1996

Light-induced charge separation between plastocyanin and the iron-sulfur clusters FA and FB in the complex of plastocyanin and photosystem I.

The light-induced electron transfer in a crosslinked complex between plastocyanin and photosystem I from spinach was studied by EPR at low temperature. Electron donation from reduced plastocyanin to P700+ was observed under illumination above a temperature of about 160 K, resulting in a second charge separation and an electron transfer from rereduced P700 to the terminal electron acceptors FA/FB. The charge-separated state Pc oxP700+ [FA/FB](2-) was found to be stable at 15 K. Implications of these results for the kinetic constants of the donation reaction and the backtransfer of electrons from reduced acceptors as well as for the structural models of the terminal acceptors are discussed.

Cross-Linking Reagents↗
PubMed1996

Binding dynamics and electron transfer between plastocyanin and photosystem I.

The mechanism of the electron transfer from the soluble protein plastocyanin to the multiprotein complex of photosystem I from spinach has been studied in detail. The two kinetic components of P700+ reduction by plastocyanin after a laser flash, showing a constant half-life of 11 microseconds and a variable half-life of the second-order reaction, respectively, are used to monitor the electron transfer from bound and soluble plastocyanin. The effect of increasing concentration of reduced plastocyanin on both of these kinetic components and the competition by oxidized plastocyanin is used to estimate the individual dissociation constants of the complex between the proteins in each of its oxidized and reduced state. The dissociation constant of oxidized plastocyanin is about six times larger than that of 7 microM found for reduced plastocyanin and purified PSI. Consistent with this result the midpoint redox potential of plastocyanin bound to photosystem I either in equilibrium with soluble plastocyanin or after cross-linking to photosystem I is found to be 50-60 mV higher than that of soluble plastocyanin. It is concluded that the driving force of the intracomplex electron transfer is decreased in favor of an optimized turnover of photosystem I. Double-flash excitation shows that oxidized plastocyanin has to leave the complex after the electron transfer before a new reduced plastocyanin molecule can bind to photosystem I. This release of oxidized plastocyanin with a half-life of about 60 microseconds limits the turnover of photosystem I. All data are consistently described by a model including the formation of a complex at a single binding site of photosystem I. Differences in the rate and binding constants are discussed with respect to the structure and the electrostatic and hydrophobic interactions stabilizing the complex as well as their modification by the membrane environment in situ.

Algorithms↗
PubMed1995

EPR, electron spin echo envelope modulation, and electron nuclear double resonance studies of the 2Fe2S centers of the 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS.

The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F. (1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz = 2.025, gy = 1.91, gx = 1.79, gav = 1.91, Em = -125 +/- 10 mV; ferredoxin-type center with gz = 2.05, gy = 1.96, gx = 1.89, gav = 1.97, Em = -200 +/- 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em = +320 mV) and the water-soluble ferredoxin (Em = -430 mV) of spinach chloroplasts, as typical representatives of the gav = 1.91 and gav = 1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.

Burkholderia cepacia↗
PubMed1995

Carotene desaturation is linked to a respiratory redox pathway in Narcissus pseudonarcissus chromoplast membranes. Involvement of a 23-kDa oxygen-evolving-complex-like protein.

The enzymic activity of phytoene desaturase in Narcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox-active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone-rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox-active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43-kDa NAD(P)H:quinone oxidoreductase, which is organized as a homodimer (23 +/- 3 kDa/subunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23-kDa protein of the oxygen-evolving complex of photosystem II, but no information about the N-terminal organization of the oxidoreductase could be obtained. During flower development, the steady-state concentration of the corresponding mRNA is up-regulated, indicating a specific function of the gene product in chlorophyll-free chromoplasts.

Amino Acid Sequence↗
PubMed1995

The 2Fe2S centres of the 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 studied by EPR spectroscopy.

The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Membrane-bound c-type cytochromes in Heliobacillus mobilis. In vivo study of the hemes involved in electron donation to the photosynthetic reaction center.

The amount of heme per photosynthetic reaction center (RC) was examined in whole cells of Heliobacillus mobilis, and a stoichiometry of 5-6 hemes c and 1-3 hemes b per RC was found. Virtually the full complement of heme was seen to be functionally connected to the pool of electron donors to the photosynthetic RC. The kinetic parameters of electron transfer between reduced c-type hemes and the photooxidized primary donor P798+ were studied in whole cells and membrane fragments. The in vivo half-times of electron donation (50% with t 1/2 = 110 microseconds, 50% with t 1/2 = 600 microseconds) were seen to slow down to half-times in the range of several and several tens of milliseconds following disruption of cells. A severe conformational alteration or a change in the identity of the donating heme is discussed. Redox titrations of the flash-induced absorption changes performed on whole cells in the presence of mediators yielded the following redox midpoint potentials: P798, Em = +240 mV; heme c553, Em = +190, +170, and +90 mV for the heme components oxidized after the first, second, and third flash, respectively. The results demonstrate that the pool of c553 hemes donating electrons to the RC is heterogeneous and that it consists of either several distinguishable cytochromes or multiheme cytochromes or both. The number of hemes reduced and the kinetics of heme rereduction after flash-induced oxidation were found to depend strongly on the degree of anaerobicity in the interior compartment of the cell. A model rationalizing the obtained results in terms of a set of differing redox components is proposed.

Bacteria↗
PubMed1993

The reaction center associated tetraheme cytochrome subunit from Chromatium vinosum revisited: a reexamination of its EPR properties.

The heme components of chromatophore membranes from the purple bacterium Chromatium vinosum have been studied by EPR. Five different heme species could be distinguished on the basis of their g values, redox midpoint potentials, and orientations of heme planes with respect to the membrane plane: gz = 2.94, Em = +10 mV, 40 degrees-50 degrees; gz = 2.94, Em = +10 mV, 0 degree; gz = 3.1, Em = +330 mV, 90 degrees; gz = 3.3, Em = 360 mV, 30 degrees; gz = 3.4, Em = 0 mV, no detectable orientation. Four of these five hemes (gz = 3.3, gz = 3.1, and 2x gz = 2.94) were ascribed to the tetraheme cytochrome subunit associated with the photosynthetic reaction center of this bacterium. Some of the results obtained have already been reported previously [Tiede, D.M., Leigh, J.S., & Dutton, P.L. (1978) Biochim. Biophys. Acta 503, 524-544] and have led to a model for the tetraheme cytochrome subunit in Chromatium which is significantly different from the three-dimensional structure of the reaction center associated subunit in the purple bacterium Rhodopseudomonas viridis. The additional data obtained in our work, however, require a reinterpretation of the previously published results. The model arrived at is in general agreement with the X-ray structure from Rhodopseudomonas viridis. A model rationalizing the detailed differences between the structure of the Rhodopseudomonas viridis cytochrome subunit and the data obtained on tetraheme subunits from other photosynthetic bacteria is presented.

Chromatium↗
PubMed1993

Color stereopsis: a model for depth reversals based on border contrast.

Color stereopsis refers to the effect of stimulus color on perceived depth of stimuli viewed binocularly. It is well established and well understood that the wavelength of a stimulus affects its perceived depth in color stereopsis by prismatic effects in human optics, with long-wavelength stimuli normally seen in front of short-wavelength stimuli. It is less well known, and not well understood, that the background of the stimulus affects depth order as well. We demonstrate experimentally, using a long-wavelength orange stimulus and a short-wavelength blue stimulus, that a change from black to white in the background at the border (a change in border contrast) of the stimulus changes the perceived depth order from orange in front of blue to blue in front of orange (a color reversal). We present a descriptive model to explain reversals in color stereopsis when stimuli are displayed on a white background and suggest that it may account for instances of color reversals that are currently unexplained or questionably explained by other models.

Adult↗
PubMed1992

The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR.

The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.

Anti-Bacterial Agents↗
PubMed1992

Characterization of an improved reaction center preparation from the photosynthetic green sulfur bacterium Chlorobium containing the FeS centers FA and FB and a bound cytochrome subunit.

A photosynthetic reaction center complex was prepared from the green sulfur bacterium Chlorobium by solubilization of chlorosome-depleted membranes with lauryl maltoside, followed by anion-exchange chromatography and molecular sieve chromatography. The purified complex was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, optical spectroscopy, and EPR spectroscopy. The major bands migrated at apparent molecular masses of 50, 42, and 32 kDa (heme-staining) and additional weaker bands at 22, 15, and 12 kDa. The isolated reaction center complex contained about 40 bacteriochlorophyll alpha molecules per primary electron donor, P840, assayed by photooxidation. It was competent in stable low-temperature photoreduction of the FeS centers FA and FB. The spectra of these acceptors and their low-temperature photochemistry in the purified complex were the same as found in intact Chlorobium membranes and similar to what had been described for photosystem I from plants. Membrane-bound cytochrome c553 copurified with the reaction center complex. A ratio of about four hemes per P840 was determined. This result indicates that cytochrome c553 that is closely associated with the reaction center is a tetraheme cytochrome, as described for some purple bacteria.

Bacteriochlorophylls↗
PubMed1991

Chloroplast Rieske Center. EPR study on its spectral characteristics, relaxation and orientation properties.

The spectral parameters, the orientation of the g tensor in two-dimensionally ordered multilayers and the relaxation properties of the Rieske center in purified cytochrome b6f complex from spinach were studied by EPR spectroscopy. A trough at g = 1.76 - 1.74 was unambiguously identified as the gx signal on the basis of the sensitivity of its field position to inhibitors and oxidized plastoquinone. In contrast to previous reports, the orientation of the chloroplast Rieske center was found to be identical to that of its cytochrome bc1 counterpart, provided that non-saturating EPR conditions were applied. Upon onset of microwave saturation, however, the orientation with respect to the ordered multilayers where the gy signal (g = 1.9) was maximal, changed drastically and was then similar to the orientation reported previously (Prince R. C., Crowder, M. S., and Bearden, A. J. (1980) Biochim. Biophys. Acta 592, 323-337). The unusually anisotropic saturation behavior of the EPR spectrum was characterized in detail, whereby hitherto controversial results on the spectral parameters of the chloroplast Rieske center could be rationalized. The observed phenomena are discussed on the basis of three tentative models which are able to qualitatively explain the effects.

Chloroplasts↗
PubMed1991

Membrane-bound cytochromes in Chloroflexus aurantiacus studied by EPR.

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em = +280 mV, 30 degrees; gz = 3.33, Em = 0 mV, 45 degrees; gz = 3.03, Em = +95 mV, 40-50 degrees and gz = 2.95, Em = +150 mV, 90 degrees. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the +95 mV and the +150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these hemes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus.

Bacteria↗
PubMed1991

Photosynthetic reaction centres: variations on a common structural theme?

From their hybrid properties, the reaction centres of green sulphur bacteria and heliobacteria seem to be the missing links between the two branches of the reaction centre family, typified by higher plant photosystem I and the purple bacterial reaction centre. This suggests that all of the diverse types of photosynthetic reaction centres have closer structural resemblances than was previously thought.

Bacterial Physiological Phenomena↗
PubMed1990

Reaction center photochemistry of Heliobacterium chlorum.

Reaction center photochemistry in Heliobacterium chlorum has been investigated by using EPR and flash absorption spectroscopy at low temperatures. The following results were obtained. At 5 K, in the presence of ascorbate, continuous illumination resulted in the formation of P798+ and a reduced iron-sulfur center designated FB (gz = 2.07, gy = 1.93, gx = 1.89). This state was stable at low temperatures, but the yield for this reaction was low, and it was estimated that it occurred only in about 3% of the centers upon the first flash. After continuous illumination of a dilute sample for 10 min, still only half of the centers attained this state. In most centers, flash excitation at 5 K produced a state which recombined with time constants of 2.5 ms (congruent to 80%) and 850 microseconds (congruent to 20%). These two phases were differently influenced by the redox state of the reaction center, indicating that two different acceptors were involved in the recombination reactions. When continuous illumination was given at 200 K, a second center, designated FA, was additionally reduced (gz = 2.05, gy = 1.95, gx = 1.90). High concentrations of dithionite resulted in the chemical reduction of FB and of most of FA; illumination at 200 K resulted in the further reduction of FA. Two triplet states were identified by EPR and optical spectroscopy. The amplitude of the narrower triplet (magnitude of D = 226 x 10(-4) cm-1) varied with the redox state of the iron-sulfur centers and was influenced by a component thought to be a quinone undergoing double reduction. It correlated with a triplet state observed by flash absorption spectroscopy showing a bleaching at 798 nm and is attributed to a triplet state formed by charge recombination in the reaction center. Its narrowness is taken as an indication of its origin on a pair of bacteriochlorophylls, and its orientation indicates an orientation of the chlorophyll ring plane perpendicular to the membrane plane. The second triplet had a wider splitting (magnitude of D = 242 x 10(-4) cm-1), did not vary systematically with redox conditions, corresponds to an optical spectrum with a maximum at 812 nm, and is not ordered in the membrane. It was thus attributed to a triplet located on a BChl g monomer in the antenna. The reaction center photochemistry in H. chlorum is comparable in many respects to that of photosystem I and green sulfur bacteria. Earlier contrasting conclusions are discussed and rationalized in light of the present results.

Bacteria↗