Chromatophores Of Chromatium Vinosum Research Articles

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.

A further study of bicarbonate effects on the ATP synthesis in chromatophores of Chromatium vinosum.

A further study of bicarbonate effects on the ATP synthesis in chromatophores of Chromatium vinosum.

The pathway of cyclic electron transport in chromatophores of Chromatium vinosum. Evidence for a Q-cycle mechanism

Chromatophores of the purple sulfur bacterium Chromatium vinosum were shown to contain a cytochrome similar to cytochrome c 1 and two b cytochromes. Cytochrome b can be accumulated in the reduced form upon illumination at an ambient redox potential of +415 mV in the presence of the electron transport inhibitors antimycin A or HOQNO. The reductions of cytochrome b, of the high-potential cytochrome c 555 and of the primary electron donor P-870 are all inhibited by myxothiazol. Dark-adapted C. vinosum chromatophores show little cytochrome b reduction on the first flash. Considerable cytochrome b reduction (1 cytochrome b:8 P-870 present) is observed on the second flash. This observation and the 1:1 stoichiometry observed between cytochrome b reduction and P-870 + reduction after the second flash support a Q-cycle model for cyclic electron flow in C. vinosum.

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. III. Basic structure of the photosynthetic unit and its relation to other bacteriochlorophyll forms.

We have performed X-ray diffraction studies on photosynthetic units of Rhodospirillum rubrum and solubilized *B800 + B890 complex from chromatophores of Chromatium vinosum, to investigate the homology of their molecular structures. The native chromatophores of Chromatium vinosum, which contain other bacteriochlorophyll forms, were examined by an X-ray diffraction technique, in order to assess the interactions between the complexes as well as the molecular structures of the bacteriochlorophyll forms. The subchromatophore particles, solubilized by Triton X-100 from cells of Chromatium vinosum, exhibit a major absorption maximum at 881 nm and a minor one at 804 nm, consisting of bacteriochlorophyll form *B800 + B890. The near-IR absorption spectrum of the particle is very similar to that of chromatophores of Rhodospirillum rubrum although the major absorption maximum is shifted slightly. The X-ray diffraction pattern of the subchromatophore particles is very similar to that of chromatophores of Rhodospirillum rubrum. Thus, the subchromatophore particles are considered to be the "photoreaction unit" of Rhodospirillum rubrum. Since the bacteriochlorophyll form, *B800 + B890, is common in the purple bacteria, it is strongly suggested that the photoreaction unit is the basic and common structure existing in the photosynthetic units of purple bacteria. Chromatium vinosum cells exhibit different near-IR absorption spectra, depending on the culture media and also on the intensity of the illumination during culture. The chromatophores from these cells give different equatorial X-ray diffraction patterns. These patterns are much broader than that of solubilized subchromatophore particles, though they have common features. Thus, the molecular structures in the photosynthetic units are different, depending on their constituent bacteriochlorophyll forms.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of cytochrome c1 in the purple sulfur bacterium Chromatium vinosum

A cytochrome-enriched preparation derived from Chromatium vinosum chromatophores has been used to demonstrate the presence of a previously undetected high potential cytochrome c in C. vinosum. The reduced cytochrome has α, β and Soret band maxima at 552–553, 523 and 422 nm respectively and a probable M r of 31 000. The cytochrome appears to have E m near +245 mV. These properties of the C. vinosum cytochrome suggest that it is similar to cytochrome c 1.

A myxothiazol-sensitive Q-binding protein isolated from Chromatium vinosum

A quinol: ferricytochrome c oxidoreductase has been isolated from chromatophores of Chromatium vinosum by two procedures, involving extraction by bile salts and methanol, respectively. The steady-state kinetics indicate a random mechanism, with a K m for 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol of 1.1 μM and for the acceptor cytochrome c 1.75 μM. The enzyme is inhibited by myxothiazol, competitively with respect to quinol, with a K i of about 2.3 μM. The protein reacts with ubiquinol produced by the succinate: Q oxidoreductase in submitochondrial particles or isolated succinate: cytochrome c reductase and can partially restore activity to myxothiazol-inhibited, antimycin-sensitive ubiquinol: cytochrome c oxidoreductase. The protein is considered to be analogous to the postulated myxothiazol-sensitive Q-binding protein in ubiquinol: cytochrome c oxidoreductase.

Regulation of electron transfer by sidedness-dependent surface pH. Dependence of the rate of cytochrome c-555 reduction on H+ concentration in the surface region on the periplasmic side of photosynthetic membranes in whole cells, spheroplasts and chromatophores of Chromatium vinosum.

Regulation of electron transfer by sidedness-dependent surface pH. Dependence of the rate of cytochrome c-555 reduction on H+ concentration in the surface region on the periplasmic side of photosynthetic membranes in whole cells, spheroplasts and chromatophores of Chromatium vinosum.

Near-infrared absorption spectra of light harvesting bacteriochlorophyll protein complexes from Chromatium vinosum.

Light harvesting (LH) bacteriochlorophyll (Bchl) protein complexes were isolated from chromatophores of Chromatium vinosum, by the combination of detergent solubilization, sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and hydroxyapatite chromatography. Addition of the absorption spectra of these complexes reproduced the absorption spectrum of chromatophores from which these complexes were derived. This result led to the conclusion that these isolated complexes retained the near-infrared absorption spectra which these complexes showed as they existed in chromatophores. Two kinds of spectrally different chromatophores were obtained under different culture conditions. One of them contained two kinds of LH Bchl protein complexes; B890 containing and B850-B800 containing complexes, and the other contained, in addition, B820-B800 containing complex. B890 containing complexes from the two types of chromatophores were spectrally similar, whereas B850-B800 containing complexes were not the same with respect to the location of absorption maxima and the content of B850. It was shown that the variation of the near-infrared absorption spectra of the chromatophores is due to not only the variation of the ratio of complexes but also the variation of the absorption spectrum of a particular complex.

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores flash-induced cytochrome b reduction

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an α-band maximum at 562 nm in the presence of antimycin. The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood. Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation. These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.

Modifiable chromatophore proteins in photosynthetic bacteria

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C.

The energy-linked carotenoid band-shift in Chromatium vinosum

Chromatium vinosum chromatophores contain an energy-linked pyrophosphatase that is insensitive to oligomycin and dicyclohexylcarbodiimide. Pyrophosphate hydrolysis produces a carotenoid band-shift similar to that resulting from illumination. The carotenoid band-shift can also be produced by a K + diffusion potential (interior positive) and the magnitude of the band shift is proportional to the membrane potential over at least a 100-fold variation in K + concentration. The light-induced carotenoid band-shift in whole cells is identical to that seen in chromatophores but K + diffusion potentials (interior positive) produce a mirror image of the light-induced band-shift. These results are interpreted in terms of chromatophores being inside-out vesicles.

Regulation of electron transfer in Chromatium vinosum chromatophores by intravesicular H+ concentration.

The possibility that electron transfer is regulated by H concentration in the inner space of chromatophores was examined in the chromatophores of Chromatium vinosum. The re-reduction of cytochrome c-555 after illumination was dependent on the pH of the suspending medium; rapid in the alkaline pH range and slow in the acidic range. A clear dependence of the half-time of the re-reduction on the intravesicular pH, estimated by the fluorescence quenching of 9-aminoacridine, was demonstrated. An almost identical dependency of the half-time on the medium pH was found in the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP). The same pH1n were also observed in the presence of valinomycin, nigericin, and valinomycin plus nigericin. The intravesicular H+ concentration, rather than the H concentration gradient across the membrane or the membrane potential, probably regulates the rate of cyclic electron transfer in Chromatium chromatophores. Under the “phosphorylating” conditions, a difference in the level of adenine nucleotides induced the regulation of electron flow. Again, a correlation of the half-reduction time and the internal H+ concentration can be clearly demonstrated. Kinetic analysis of the reduction of cytochrome c-555 indicated the presence of two electron-donating pools. The rapid electron donation was pH-sensitive and the slower electron donation from a large secondary pool was pH-insensitive and probably diffusion-controlled.

Dichroism of bacteriochlorophyll in chromatophores of photosynthetic bacteria.

The dichroism was measured in films of air-dried and, consequently, flattened chromatophores of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum. The values (deltaA/A) of dichroism in C. vinosum were found to be -1.05 at 590 nm and 0.75 in the near infrared region. The values of dichroism in R. sphaeroides were -0.70 at 590 nm and 0.80 at 870 nm. The values of dichroism in R. rubrum were -1.45 at 590 nm and 0.97 at 870 nm.

Differentiation of the photosynthetic apparatus of Chromatium vinosum, strain D

Spectral changes in the near infrared absorption region of chromatophores of Chromatium vinosum, strain D, were analyzed. Spectral changes dependent in growing cultures on temperature (33° C and 39° C, respectively) were compared to artificial changes which take place under the influence of the detergent Triton X-100. Like addition of Triton X-100 to chromatophores, transfer of cells from 39° C to 33° C leads to the reversible formation of an absorption band at 820 nm at the expense of a band at 850 nm. But in contrast to the influence of Triton X-100, chromatophores isolated from cells grown at 39° C (39° C chromatophores), contain on an identical bacteriochlorophyll basis, higher amounts of pigment absorbing at 880 nm and lower amounts of pigment absorbing at 800 nm than found in 33° C chromatophores. Triton X-100 treatment does not influence the production of light induced absorbance changes characteristic of photochemical reaction centers; it does, however, change absorption spectra of a subchromatophore fraction which does not exhibit the known light dependent reaction center responses. The spectral properties of this fraction are altered with Triton X-100 in a manner comparable to whole chromatophores, i.e. translocation of the 850 nm band to 820 nm. Subchromatophore fractions isolated from 33° C chromatophores, which display an absorption band at 820 nm show in the presence of menadione a light induced absorbance change at 835 nm. Subchromatophore fractions from 39° C chromatophores, which display no absorbance at 820 nm show a light induced adsorbance change at 835 nm only after addition of Triton X-100. Spectral changes in growing cultures are accompanied by changes in the fluorescence emission spectra of isolated chromatophores. While 33° C chromatophores exhibit a major fluorescence emission band at 929 nm plus a minor band at 890 nm 39° C chromatophores exhibit only one band at 937 nm.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores: characteristics in the presence of o-phenanthroline.

Delayed fluorescence from bacteriochlorophyll in the chromatophores of Chromatium vinosum, a photosynthetic purple sulfur bacterium, was studied in the presence of o-phenanthroline (o-phen) under intermittent illumination. Re-reduction of the photooxidized reaction center bacteriochlorophyll (P+) in the dark interval was accelerated by o-phen. This effect was attributed to the return of electrons trapped in the primary electron acceptor (A) to P+. In the presence of o-phen, the time course of the decay of delayed fluorescence was not coincident with that of the re-reduction of P+. The delayed fluorescence was somewhat intensified at the early stage (within 30 ms) of relaxation in the dark period. Prolonged illumination (longer than 20 ms) or uncouplers such as carbonylcyanide m-chlorophenylhydrazone (CCCP) or valinomycin plus nigericin decreased the intensity of delayed fluorescence and suppressed the stimulation of delayed fluorescence at the early stage. Delayed fluorescence from reaction center-rich subchromatophore particles decayed with a time course identical to that of the reduction of P+ and was not affected by CCCP, in the presence of o-phen. The intensification at the early stage in the chromatophores can be interpreted in terms of charge separation between pairs of P and A, primary electron donor and acceptor molecules, oriented perpendicular to the intact chromatophore membrane, the effect decreasing in parallel with the recombination of P+ and A-.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores was studied at room temperature and under intermittent illuminations. The decay of delayed fluorescence was constituted of two components; a fast component decayed with a half time of about 8 ms, a slow one decayed in parallel with the reduction of photooxidized bacteriochlorophyll ( P +) with a half time of 100–200 ms. The biphasic decay of delayed fluorescence indicated that a rapid equilibrium was established between the primary electron acceptor and the secondary acceptor. In the presence of o-phenanthroline, the time course of the decay of delayed fluorescence was identical with that of the reduction of P + in reaction center-rich subchromatophore particles, although they did not necessarily coincide with each other in “intact” chromatophores. The intensity of the slow component was increased and the decay was accelerated at basic pH values. Reagents that dissipate the proton gradient across the chromatophore membranes such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and nigericin accelerated the decay of the slow component. These effects are probably resulting from changes in internal pH of chromatophore vesicles. Reagents that dissipate the membrane potential such as CCCP and valinomycin decreased the intensity.

Identification of ubiquinone as the secondary electron acceptor in the photosynthetic apparatus of Chromatium vinosum

Extracting Chromatium vinosum chromatophores with light petroleum destroys their ability to perform photochemistry on the second of two closely-spaced actinic flashes, without affecting photochemistry on the first flash. Extraction also increases the likelihood of a back-reaction in which an electron returns from the primary electron acceptor directly to P 870. These effects probably reflect the removal of a secondary electron acceptor. Extraction does not appear to interfere with the primary photochemical reaction. Reconstituting the extracted chromatophores with the lipid extract or with pure ubiquinone (Q) completely reverses the effects of the extraction. Chromatography of the lipid extract shows that Q is the only active material that it contains in detectable quantity. These observations support the conclusion that Q is the secondary electron acceptor. Piericidin A, certain alkyl-substituted quinolinequinones, and a substituted 4,7-dioxobenzothiazole inhibit electron transfer between the primary and secondary acceptors. The sensitivity to these inhibitors, and the participation of Q and non-heme iron suggest that the secondary electron-transfer reaction resembles the reactions catalyzed by respiratory dehydrogenases. The proton uptake that follows flash excitation does not seem to be tightly linked to the reduction of the secondary electron acceptor. It still occurs (though with decreased amplitude) in extracted chromatophores, and even in the presence of inhibitors of the secondary electron-transfer reaction.

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P 870 + after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P 870 itself, nor the oxidation of cytochrome c-552 by P 870 +. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P 870 oxidation. The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes. Previous work has shown that the photooxidations of P 870 and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.