Chromatium Chromatophores Research Articles

Regulation of nitrogenase activity by covalent modification in Chromatium vinosum.

Nitrogenase in Chromatium vinosum was rapidly, but reversibly inhibited by NH4+. Activity of the Fe protein component of nitrogenase required both Mn2+ and activating enzyme. Activating enzyme from Rhodospirillum rubrum could replace Chromatium chromatophores in activating the Chromatium Fe protein, and conversely, a protein fraction prepared from Chromatium chromatophores was effective in activating R. rubrum Fe protein. Inactive Chromatium Fe protein contained a peptide covalently modified by a phosphate-containing molecule, which migrated the same in SDS-polyacrylamide gels as the modified subunit of R. rubrum Fe protein. In sum, these observations suggest that Chromatium nitrogenase activity is regulated by a covalent modification of the Fe protein in a manner similar to that of R. rubrum.

Interaction between antimycin and cytochrome b560 in Chromatium chromatophores--The red shift of the absorption spectrum and photoreduction of cytochrome b560 in the presence of antimycin

In the presence of dithionite, which reduced all the cytochromes present in Chromatium chromatophores, the addition of antimycin caused a shift (1-2 nm) of the α band in the absorption spectrum of reduced cytochrome b560 toward longer wavelength. The difference spectrum (antimycin-added-minus-no addition) had a trough at 556 nm, a peak at 564 nm and an isosbestic point at 561 nm. The locations of the trough, peak and isosbestic point did not depend on the concentration of antimycin, but the amount of shifted cytochrome b560 increased as the concentration of antimycin increased. As reported previously, a single-turnover flash reduced cytochrome b560 in the presence of antimycin. The extent of the reduction depended on the concentration of antimycin. Both titrations of the red shift and the photoreduction of cytochrome b560 with antimycin fitted a first-order sigmoid curve with a half-maximum value at 5 × 10(-7) m of antimycin concentration. The results indicated that antimycin molecules interacted with, or were bound to, cytochrome b560 molecules in the ratio of one to one with an apparent dissociation constant of 3.5-7.3 × 10(-7) m.

Light-induced blue shift of the carotenoid spectrum in chromatophores of Chromatium vinosum strain D

In Chromatium chromatophores, the response of part of the carotenoid complement to a light-induced membrane potential is a shift to the blue of its absorption spectrum, as indicated by the characteristics of the light-minus-dark difference spectrum. The spectrum in the dark of the population of carotenoid which responds to a light-induced membrane potential is located at least 1–2 nm to the red in comparison to the total carotenoid absorption. The results indicate that the proposed permanent electric field affecting the responding population has a polarity with respect to the chromatophore membrane opposite to that in Rhodopseudomonas sphaeroides chromatophores. The carotenoid absorption change interferes seriously with measurements of cytochrome c-555 redox changes at its α band.

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.

Dual roles of ubiquinone as primary and secondary electron acceptors in light-induced electron transfer in chromatophores of Chromatium D.

Dual roles of ubiquinone as primary and secondary electron acceptors in light-induced electron transfer in chromatophores of Chromatium D.

Nature of photochemical reactions in chromatophores of Chromatium D III. Heterogeneity of the photosynthetic units

The effect of isooctane extraction on photooxidation of c-type cytochromes was investigated in Chromatium chromatophores. Photooxidation of cytochrome c-555 was not affected by isooctane-extraction except that the dark recovery was accelerated. Photooxidation of cytochrome c-552 was abolished by thorough extraction of ubiquinone-7, but the quantum yield of the cytochrome photooxidation remained unchanged until 90|X% of the total ubiquinone was extracted. The photooxidation of cytochrome c-552 was recovered by the addition of ubiquinone-7 but not by menaquinone. A dark incubation of sufficient length was needed for maximal quantum yield of cytochrome c-555 photooxidation in the presence of 30 mM ascorbate. It is proposed that there are two types of photosynthetic units (or associations of molecules involved in the primary redox reactions) in Chromatium chromatophores. The combinations of primary electron donor-reaction center chlorophyll-primary electron acceptor may be cytochrome c-552-P890-ubiquinone in one type and cytochrome c-555-P890-X in another.

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome α-band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll). The b-type cytochrome in Chromatium has an α-band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an α-band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV. Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2- n-heptyl-4-hydroxyquinoline- N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na 2S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.

Nature of photochemical reactions in chromatophores of Chromatium D. II. Quantum yield of photooxidation of cytochromes in Chromatium chromatophores

Initial rates of the light-induced absorption decrease in Chromatium chromatophores due to the oxidation of cytochromes were measured under various experimental conditions. The initial rate in the presence of 10 mM potassium ferrocyanide and 50 μM potassium ferricyanide was about one-half to two-thirds of that in the presence of 30 mM ascorbate or in a medium with a redox potential ( E h) of − 78 mV. Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at E h values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4. The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed.

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10 4. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential ( E m) of that carrier should be a function of the solution ionic strength ( I). of that carrier should be a function of the solution ionic strength ( I). The E m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases. The E m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane. The E m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.

Fast changes of enthalpy and volume on flash excitation of Chromatium chromatophores

We have used a capacitor microphone transducer to measure volume changes in suspensions of Chromatium chromatophores 100 μs to 20 ms after flash excitation. Volume changes in photochemical reactions can arise both from volume differences between reactants and products, and from enthalpy changes which heat or cool the solution. By measuring the volume changes at two temperatures, one can resolve the total changes into their two components. In Chromatium, light-driven electron transfer from cytochrome C555 to the primary or secondary electron acceptors causes a contraction of approx. 33 Å 3 per electron transferred. The relaxation of the initial volume change is altered by the presence of gramicidin, valinomycin, o-phenanthroline, or phosphate. From the effects of these agents, we conclude that the volume change is localized at the photochemical reaction center, and that part of the relaxation involves transfer of a proton from the solvent to the chromatophores. Electron transfer from cytochrome C555 to the primary or secondary electron acceptors does not cause a significant enthalpy change. We conclude that the free energy increase that accompanies this reaction is due to a negative entropy change.

LUMINESCENCE IN PHOTOSYNTHETIC BACTERIA*

Abstract— The behavior of glow curves and of ferricyanide‐induced luminescence in photo‐synthetic bacteria is consistent with a model in which light is emitted when electrons return to oxidized reaction center chlorophyll from the primary acceptor. A number of observations can be explained if it is assumed that the chromatophore membrane potential provides part of the activation energy for the process. The model is tested by inducing light emission by the generation of potassium diffusion potentials in preilluminated Chromatium chromatophores.

Spectral and photochemical properties of subchromatophore fractions derived from carotenoid-deficient Chromatium by Triton treatment

Triton treatment of chromatophores of carotenoid-deficient Chromatium followed by density-gradient centrifugation led to a separation into three subchromatophore fractions. Unlike the case with chromatophores of regular Chromatium, Triton releases about 1 3 of the total bulk bacteriochlorophyll into one fraction (designated G, for green) whose major absorption-band maximum is at 780 nm. One fraction (H, for heavy) absorbs at 805 and 885 nm, with an absorbance ratio A 885 nm A 805 nm between 1.5 and 2; another fraction (L, for light) absorbs at 805 nm and has a shoulder at 825 nm. The absorption and fluorescence emission spectra of the three fractions at room temperature and 77°K indicate that the different bacteriochlorophyll forms are efficiently separated by Triton treatment. The reaction center P890 is concentrated exclusively in the H-fraction, at a level of 5–7% of the bulk bacteriochlorophyll. The solubilized bacteriochlorophyll absorbing at 780 nm can be totally and irreversibly bleached by 5 mM ferricyanide. The other bacteriochlorophyll forms in the H- and L-fractions are also irreversibly bleached by ferricyanide to variable extents. P890 is the only component that can be re-reduced by ascorbate after ferricyanide oxidation. The P890 content estimated by reversible chemical bleaching agrees well with that obtained by reversible light bleaching. The different bacteriochlorophyll forms, with the exception of the 780-nm absorbing form, are relatively stable toward light bleaching. Again, only P890 is reversibly bleached by light. Cytochromes-555 and -553 are distributed in both the H-and L-fractions, but not in the solubilized-bacteriochlorophyll G-fraction. However, only cytochromes in the H-fraction which contains all of the P890 can undergo coupled oxidation. Excitation with 20-nsec ruby-laser pulses shows that cytochrome-555 can be oxidized in 2–3 μsec by photooxidized P890, indicating that necessary conformation for rapid electron transport is retained in the subchromatophore particles. The data on fractionation and redox reactions obtained here, together with direct kinetic measurements recently reported in the literature lend further support to the view that oxidation of these two cytochromes is mediated by the same reaction center, P890.

A low potential photosystem in Chromatium D

A low potential light reaction in chromatophores of Chromatium D has been studied by means of oxidation—reduction potential titrations of laser-induced absorbance changes at 435 nm and 410 nm. This reaction, operating at potentials which are low enough to inactivate the P883 (P435) reaction center, has an apparent E m of −145 mV and is attenuated as the potential is lowered with an E m of −318 mV (n = 1). Its spectrum at −260 mV has a peak at 424 nm (compared with peaks at 423 nm and 432 nm for P435 at +415 mV), and its rise half-time appears to be around 75 nsec (compared with <50 nsec for P435). Although this system may represent either a new reaction center or a different form of P883, it is apparent that thermodynamically it is able to donate electrons to NAD +.

Photooxidation of cytochromes in reaction center preparations from Chromatium and Rhodopseudomonas viridis

In a reaction center preparation from Rhodopseudomonas viridis, which contains as its only bacteriochlorophyll a single reactive trimer, two bound cytochromes under-go photooxidation. Cytochrome C558 photooxidation occurs at redox potentials above +100 mV, with a half-time of approx. 1 μsec. Cytochrome C 553 photooxidation occurs at potentials below o mV, with a rate that is presently too fast to measure. Similarly, a subchomatophore fraction from Chromatium exhibits photooxidation of bound cytochrome C555 with a half-time of 2.3 μsec at potentials above +100 mV, and cytochrome C552 with a half-time of 1.1 μsec at potentials below o mV. The changeover from one cytochrome to the other fits a one-electron titration curve with E m = +25 mV, which is independent of the pH. At all potentials, the rate of P883 + (P870+) reduction is the same as that of cytochrome oxidation. Measurements of P883+ reduction at both 882 and 785 nm allow the conclusion that this reactive bacteriochlorophyll species oxidizes both cytochromes. The similarities between the light-induced reactions in Chromatium and Rps. viridis suggest that a general feature of bacterial photosynthesis may be the oxidation of both high- and low-potential cytochromes by a single photochemical system. The cytochrome oxidation kinetics in the Chromatium fraction are essentially identical with those in Chromatium chromatophores; however, the reaction between the primary and secondary electron acceptors is slower by a factor of 1×10 3. The purified material appears to lack the secondary acceptor, Y. N-Methylphenazonium methosulfate (PMS) or methylene blue can replace Y in a reaction which, unlike the in vivo reaction, is insensitive to 1,10-phenanthroline.

Nature of photochemical reactions in chromatophores of Chromatium D. I. Effects of isooctane extraction on the photochemical reactions of P 890 and ubiquinone in chromatophores of Chromatium D

1. 1. Effects of various experimental conditions on the light-induced reduction of ubiquinone in the chromatophores of Chromatium D were investigated. 2. 2. Addition of Na 2S 2O 3 or malate and change of aero- to anaerobiosis did not affect the reactions of ubiquinone, thus indicating the lack of non-cyclic electron paths in the isolated chromatophores. 2- n-Heptyl-4-hydroxyquinoline- N-oxide and antimycin A slowed down the dark reoxidation of photoreduced ubiquinone, yet still suppressed slightly the magnitude of photoreduction of ubiquinone. Evidence was obtained that indicated that the observed absorption change of ubiquinone is caused by a cyclic electron flow in the chromatophores. 3. 3. Extraction of chromatophores with isooctane diminished the light-induced absorption changes in the ultraviolet and near-infrared regions, which were recovered by readdition of ubiquinone. Extraction of less than 80% ubiquinone was without effect, and readdition of only 5% of the endogenous ubiquinone sufficed to cause a noticeable recovery both with respect to the changes of ubiquinone and that of P 890 in the extracted chromatophores. 4. 4. These facts indicate that a special small portion of endogenous ubiquinone characterized by a strong affinity towards combination with the photochemical reaction site is participating in the cyclic electron transport, which makes the primary photochemical reaction in the chromatophores. 5. A light-induced decrease occurred at 283 nm that survived extensive extraction with isooctane, probably ascribable to a redox reaction of an unidentified substance contained in the chromatophores.

Fast membrane H+ binding in the light-activated state of Chromatium chromatophores.

A fast and sensitive single‐beam spectrophotometer and a variety of flash and steady‐state illumination sources permit the resolution of bromcresol purple absorbancy changes from background absorbancy changes of Chromatium chromatophores, and identification of these fast absorbancy changes with rapid H+ binding by the chromatophores. A saturating light flash gives a rate several hundred times faster than that obtained in continuous light, the maximal rate being about 50 H+/chlorophyll μsec, with a magnitude of the H+: chlorophyll ratio of 1:30 and a duration for the H+ binding reaction, measured by absorbancy changes of bromcresol purple, of 2.2 msec (half‐time, 800 μsec for a 200 μsec flash). A single turnover flash (10 μsec) yields an H+: chlorophyll value of 1:100, with a duration of the H+ change of 800 μsec and a half‐time of 400 μsec. A double‐flash technique shows the recovery for the fast H+ reaction to have a half‐time of 60 msec. The effects of pH, temperature, uncouplers, and inhibitors are also reported.While the hydrogen ion binding would appear to be closely associated with the reduction of an electron acceptor, Y, particularly on the basis of the similar amounts of the two substances (roughly one each per hundred chlorophyll) the reduction time for Y of 27 μsec is over ten‐fold shorter than the H+ binding time in single flash experiments. In addition, the pH sensitivities and activation energies of the two reactions differ.Thus, either an unidentified electron acceptor is involved in the fast H+ changes, or there is no direct relationship between electron transport and H+ binding. If the latter is the case, we propose that electron transport through the photosynthetic center produces in 800 μsec a state of the membrane (a “membrane Bohr effect”) in which hydrogen ion binding is considerably enhanced.

Fast spectrophotometric measurement of H+ changes in Chromatium chromatophores activated by a liquid dye laser.

The fast changes in hydrogen ion concentration which follow photo-activation of the bacteriochlorophyll complex of Chromatium have been measured by observing absorbance changes of the pH indicator bromcresol purple using a ruby-pumped dye laser. This acts as an optical frequency converter with an output pulse corresponding to an absorption peak of the chromatophore. The changes in H+ binding represent a sort of “membrane Bohr effect”.

The reaction between primary and secondary electron acceptors in bacterial photosynthesis

Following a 20-nsec actinic flash, which causes oxidation of P870 and cytochrome C422, Chromatium chromatophores enter a refractory state. While the chromatophores are in this state, a second flash does not cause further oxidation of P870 or cytochrome C422. The quanta of the second flash are wasted as fluorescence (and heat); apparently they do not energize an alternative photochemical reaction. The refractory state probably reflects the accumulation of the primary electron acceptor in a reduced form. By following the reappearance of the capacity for photochemistry, one can measure the kinetics of electron transfer between the primary electron acceptor and the secondary agent which reoxidizes it. In Chromatium chromatophores, this process requires about 60 μsec to proceed half-way to completion at pH 7, and 80 μsec at pH 8. The rate of the reaction increases with decreasing pH, but not in direct proportion to the proton concentration. It increases with temperature, with an E a of about 8.3 kcal/mole. The kinetics are approximately second order in the concentration of the reduced acceptor.

Cytochrome photooxidations in Chromatium chromatophores: Each P870 oxidizes two cytochrome C422 hemes

A single, 20-nsec actinic flash oxidizes all of the P870 in Chromatium chromatophores, but only about one half of the cytochrome C422. A second flash, 1–10 msec later, oxidizes most of the remaining cytochrome. The cytochromes which undergo oxidation on the first and second flashes are indistinguishable with respect to their absorption spectra, their kinetics of oxidation and reduction, and their response to N-methylphenazonium methosulfate (PMS) or continuous actinic illumination. The effect of PMS is to increase the total amount of cytochrome C422 which is in the reduced form in the dark, and which is available for photooxidation. The conclusion is that each P870 reaction center is responsible for the oxidation of two C422 hemes.

Reversible interconversion of B850 and B810 in chromatophores of Chromatium D as induced with detergents

The absorption changes of bacteriochlorophyll in the chromatophores of Chromatium D were investigated. Addition of non-ionic and cationic detergents and alcohols to the chromatophore suspension caused a conversion of B850 to B810. A similar result was obtained with treatments which reduce the water content of the chromatophores ( e.g., drying, treatment with higher concentrations of neutral salts or glycerol). On reducing the concentration of the detergents and alcohols in the chromatophore suspension by dialysis, the absorption spectrum of the chromatophores completely returned to its original state. A similar recovery of absorption spectrum was obtained by wetting the dried films of chromatophores or by diluting the salts or glycerol solution with water. No significant changes occurred in B890 during these interconversions. These treatments also induced reversible changes in the fluorescence emission spectrum of bacteriochlorophyll and the action spectrum of photo-induced oxidation of c-type cytochrome in the chromatophore.