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.     

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.     

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.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.     

Thermal stabilities of membrane-bound,solubilized, and artificially immobilized hydrogenase from Chromatium vinosum

Thermal inactivation (at 65 °C) of both membrane-bound and solubilized hydrogenase from Chromatium vinosum has been studied. Membrane binding greatly increases thermostability relative to the solubilized enzyme. Covalent attachment (but not simple adsorption) of the solubilized enzyme to ω-NH₂-alkyl-agaroses sharply increases thermal stability, which almost attains that of membrane-bound hydrogenase. It appears that there are at least two requirements for such stabilization—the enzyme should be in a hydrophobic medium and should be firmly bound to a hydrophobic support.     

Magnetic interaction of nickel(III) and the iron-sulphur cluster in hydrogenase from Chromatium vinosum

Upon partial reduction of hydrogenase from Chromatium vinosum with ascorbate plus phenazine methosulphate, EPR signals due to Ni(III) and a [3Fe-xS] cluster appear simultaneously and with equal intensities. Since the intact enzyme shows no $\text{S =}\text{1}\text{2}$ signals, it is concluded that Ni(III) and a [4Fe-4S]³⁺ cluster interact magnetically in such a way as to prevent the detection of the two paramagnets as individual $\text{S =}\text{1}\text{2}$ systems. This interaction is thought to be the origin of a signal in which Fe is involved and which is not due to an $\text{S =}\text{1}\text{2}$ system (Albracht, S.P.J., Albrecht-Ellmer, K.J., Schmedding, D.J.M. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 330–334). A variable fraction of the enzyme preparation shows signals due to Ni(III) and a [3Fe-xS] cluster with equal intensities without any further treatment. These are thought to be derived from irreversibly inactivated enzyme molecules. The enzyme contains no selenium.     

Studies of the function of the membrane-bound iron-sulfur centers of the photosynthetic bacterium Chromatium vinosum

Chromatophores from the photosynthetic bacterium, Chromatium vinosum, have been prepared which photoreduce NAD⁺ with either succinate or reduced dichlorophenolindophenol as electron donors. NAD⁺ reduction is inhibited by uncouplers as well as inhibitors of cyclic photophosphorylation. These chromatophores contain several bound iron-sulfur centers which have been detected by low-temperature EPR spectroscopy. One center, having a g 2.01 EPR signal in the oxidized state, has E_m7.5 = +50 mV and is partially reduced by succinate in the dark. Three iron-sulfur centers having g 1.93 EPR signals have been resolved by redox titration, and the E_m7.5 values of these centers are ?50, ?175 and ?250 mV, respectively. Studies of the involvement of these centers in electron transfer from donors to NAD⁺ have indicated that the center with E_m = ?50 mV is succinate reducible in the dark and appears to be analogous to center S-1 of succinic dehydrogenase in other systems. An additional g 1.93 iron-sulfur center can be photoreduced in the presence of electron donors and this reduction is inhibited by uncouplers. The possible role of the two low-potential iron-sulfur centers in relation to the dehydrogenases functioning in NAD⁺ reduction is considered.     

The role of soluble cytochrome c-551 in cyclic electron flow-driven active transport in Chromatium vinosum

Spheroplasts have been prepared from the photosynthetic purple sulfur bacterium Chromatium vinosum by lysozyme plus ethylenediaminetetraacetic acid treatment. These spheroplasts are able to take up alanine in the light, but light-dependent alanine uptake is lost upon subsequent washing of the spheroplasts. The observations that alanine uptake driven by a potassium plus valinomycin-induced membrane potential (outside positive) is not affected by washing and that light-dependent alanine uptake can be restored by addition of the supernatant from washing suggest that a soluble electron carrier is lost during washing. Light-dependent alanine uptake in washed spheroplasts could be restored by addition of C. vinosum cytochrome c-551. Other soluble electron carriers from C. vinosum (high-potential iron protein, cytochrome ‘f’, cytochrome c′ and the flavocytochrome c-552) did not restore alanine uptake nor did a variety of other soluble electron carrier proteins from other organisms. These results suggest that cytochrome c-551 functions as an electron carrier in the cyclic electron transfer chain of C. vinosum. Mitochondrial cytochrome c (equine heart) and cytochrome c-551 from Pseudomonas aeruginosa were highly effective in restoring light-dependent alanine uptake in washed spheroplasts, making it likely that C. vinosum cytochrome c-551 is related by evolution to the same cytochrome c family as these other two c cytochromes.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

1. The cytochromes of chromatophores from photosynthetically grown Rhodopseudomonas capsulata have been characterised both spectrally, using the carotenoid free mutant Ala Pho⁺, and thermodynamically, using the technique of redox titrations. Five cytochromes were present; two cytochromes b, E′₀ = 60 mV at pH 7.0; and three cytochromes c, E′₀ = 340 mV, $\text{E}\text{t}\text{?}{}_0\text{= 120}\text{mV}$, E′₀ = 0 mV at pH 7.0.2. Redox titrations at different values of pH indicated that the mid point potentials of all the cytochromes varied with pH over some parts of the range between pH 6 and 9, with the possible exception of cytochrome c₃₄₀.3. The effects of succinate and NADH on the steady state reduction of the cytochromes are reported. Succinate could reduce cytochromes c₃₄₀, c₁₂₀ and b₆₀; NADH could reduce cytochromes c₃₄₀, c₁₂₀, b₆₀ and b_?25. Cytochrome c₀ could be reduced by dithionite but not by the other substrates tested.     

Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum.This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1].After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.3}\text{ms}$). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c⁺-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c⁺-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A.Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution.Analysis of the reduction kinetics of c⁺-551 in the absence and presence of external donors suggests that c⁺-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c⁺-551 (k = 6 s^?1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.     

The architecture of Rhodobacter sphaeroides chromatophores

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc₁ complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc₁ complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc₁ complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Arsenylation of nucleoside diphosphates in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores catalyze the formation of ADP-arsenate during illumination with ADP, Mg²⁺ and arsenate. The reaction was measured with (1) firefly luciferase, (2) a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase, and (3) a glass electrode. ADP-arsenate hydrolyzed spontaneously with rate constants ranging from 14 to 43 min^?1. Magnesium, arsenate and phosphate accelerated hydrolysis of ADP-arsenate. From a comparison of the three methods, with ADP as the substrate, it is estimated that φ_R (i.e., the ratio between the quantum yields of ADP-arsenate and ATP for light emission from luciferase) is 0.19–0.23. Furthermore, arsenylation rates were 46–52% of phosphorylation rates in experiments with 30 μ M ADP and 0.8 mM arsenate or phosphate. Similarly, the V_app for arsenylation of GDP or IDP was 47–59% of the V_app for phosphorylation during measurements in the presence of 1 mM arsenate or phosphate. The K_app(GDP) was higher during arsenylation than during phosphorylation; the K_app(IDP) was about the same during arsenylation as during phosphorylation. It is suggested that a shift in the equilibrium of substrates and products on the enzyme, toward hydrolysis, is the main cause of the relatively low arsenylation rates.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

Membrane-potential- and surface-potential-induced absorbance changes of merocyanine dyes added to chromatophores from Rhodopseudomonas sphaeroides

(1) Three analogs of merocyanine dyes added to suspensions of chromatophore vesicles showed absorbance changes responding to the change in surface potential induced by salt addition and to the change in membrane potential induced by illumination. (2) The extent of the light-induced absorbance changes of the dyes was linearly related, in the presence and absence of uncouplers, to that of carotenoid spectral shift which is an intrinsic probe of the intramembrane electric field. (3) Comparison of the merocyanine absorbance changes induced by salt addition with those induced by illumination indicated that the surface potential change in the outer surface of chromatophore membranes during illumination was very small. (4) Judging from the spectra of these absorbance and from the low permeabilities of the dyes to membrane, the absorbance change are attributed to change in distribution of the dyes between the medium and the outer surface region in chromatophore membranes. The extent of the light-induced absorbance changes of merocyanine dyes depended on the salt concentration of the medium. The types of dependence were different among three merocyanine analogs. This is explained by the mechanism mentioned above assuming appropriate parameters. It is suggested that, under continuous illumination, an equilibrium of the electrochemical potential of H⁺ is reached between the bulk aqueous phase and the outer surface region in the membrane where the merocyanine dyes are distributed.     

The relation between membrane ionic current and ATP synthesis in chromatophores from Rhodopseudomonas capsulata

(1) Under conditions in which membrane potential (Δψ) was the sole contributor to the proton-motive force, the steady-state rate of ATP synthesis in chromatophores increased disproportionately when Δψ was increased: the rate had an approximately sixth-power dependence on Δψ. (2) Simultaneous measurements showed that the dissipative ionic current (J_DIS) across the chromatophore membrane had a related dependence on Δψ, i.e., the membrane conductance increased markedly as Δψ increased. (3) For comparable Δψ values, J_DIS was greater in phosphorylating than in non-phosphorylating chromatophores. For comparable actinic light intensities, Δψ was smaller in phosphorylating than in non-phosphorylating chromatophores. (4) At either low pH or in the presence of venturicidin, oligomycin or dicyclohexylcarbodiimide to inhibit ATP synthesis, J_DIS was substantially depressed, particularly at high Δψ. Even under these conditions the membrane conductance was dependent on Δψ. (5) Also in intact cells, J_DIS was depressed in the presence of venturicidin. Points 1–5 are interpreted in terms of a Δψ -driven H⁺ flux through the F₀ channel of the ATPase synthase. The high-power dependence of the F₀ conductance on Δψ determines the dependence of the rate of ATP synthesis on Δψ. The Δψ -dependent conductance of F₀ dominates the electrical properties of the membrane. In chromatophores the ionic current accompanying ATP synthesis was more than 50% of the total membrane ionic current at maximal Δψ. (6) The rate of cyclic electron transport was calculated from J_DIS. This led to an estimate of 0.77 ± 0.22 for the $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio and of 3.5 ± 1.3 for the $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratio. (7) Severe inhibition of the electron-transport rate by decreasing the light intensity led to an almost proportionate decrease in the rate of ATP synthesis. The chromatophores were able to maintain proportionality by confining electron-transport phosphorylation to a narrow range of Δψ. This is a consequence of the remarkable conductance properties of the membrane.     

Light-induced spectral changes of carotenoids in chromatophores of Rhodopseudomonas sphaeroides

Carotenoid difference spectra were recorded of chromatophores of Rhodopseudomonas sphaeroides energized with low light intensities versus chromatophores under dark conditions. The amplitudes of the peaks and troughs were dependent on the light intensities and the duration of illumination, but the shape of the difference spectra remained constant. Sharp isosbestic points were found at 515, 500, 481, 465, and 452 mm. When potassium-valinomycin-induced diffusion potentials (inside negative) were imposed on the chromatophores “mirror images” of the light-induced difference spectra were recorded with the same isosbestic points and the same positions but different relative amplitudes of the peaks and troughs. The results are explained in terms of changes in the shape of the vibrational splittings of the carotenoid main electronic transition. This could be the result of changes in the fluidity of the environment of the carotenoids upon energization of the membrane. Prolonged periods of illumination with low or high light intensities resulted in irreversible changes of the difference spectra. Short periods of illumination with high light intensities resulted in reversible elevations of the baseline and red shifts of peaks, troughs, and baseline crossings.