Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes.

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associtated with planas azolectin membrane, ptoteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgC1 electrodes and voltmeter. The direction of the electric field inproteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.     

Absorption changes of carotenoids and bacteriochlorophyll in energized chromatophores of Rhodospirillum rubrum

The energization of Rhodospirillum rubrum chromatophores by the light, ATP, PP_i, by dark electron transfer via energy-coupling sites of the redox chain, by the combination of KC1 and valinomycin causes absorption changes of carotenoids and bacteriochlorophyll. These changes due to the absorption-band shifts of the pigments are sensitive to the uncoupler p-trifluoromethoxycarbonyl cyanide phenylhydrazone (FCCP) but not to the combination of KC1 and nigericin, which abolishes fluorescence changes of atebrin. Dithionite and ferricyanide depress the light-induced absorption changes of bacteriochlorophyll but have no inhibitory effect on the PP_i-induced changes. Analysis of bacteriochlorophyll absorption changes in the infra-red region shows that the photooxidation of bacteriochlorophyll reaction centers with the negative peak in the region of 890 nm is accompanied by red and blue shifts of bacteriochlorophyll absorption bands. These shifts are due to a transmembrane electrochemical gradient of H⁺ and a local electric field arising as a result of oxidation of the reaction centers. It appears that the superposition of the (1) red shift which is characterized by negative and positive peaks at 865 and 895 nm, respectively, and (2) photobleaching of bacteriochlorophyll reaction centers in the region of 890 nm cause overall absorption changes with the negative peak at 865 nm.     

Photoinactivation of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores

Preillumination of Rhodospirillum rubrum chromatophores with strong, far-red light in the presence of phenazine methosulfate under non-phosphorylation conditions results in a selective, irreversible inactivation (typically about 70%) of photophosphorylation and of uncoupler-stimulated dark ATPase. The time course of the photoinactivation is similar to the light-on kinetics of the light-induced proton uptake in the absence of ADP. Only little photoinactivation occurs when the uncoupler carbonyl cyanide m-chlorophenyl hydrazone is present or when phenazine methosulfate is absent during the preillumination, indicating that the reaction occurs only when the membrane is energized.

Phosphorylation conditions offer a practically complete protection against the photoinactivation. Inorganic phosphate, Mg²⁺ or ADP do not provide a significant protection against the photoinactivation, nor does ATP. The pH-dependence of the reaction(s) leading to photoinactivation may indicate that a partial reaction of the photophosphorylation process (perhaps only a conformational change of the coupling factor) precedes the photoinactivation.     

A study on the membrane potential and pH gradient in chromatophores and intact cells of photosynthetic bacteria

Generation of membrane potential (Δψ) and transmembrane pH difference (ΔpH) was studied in PP_i-energized chromatophores of Rhodospirillum rubrum by means of measurements of carotenoid and bacteriochlorophyll absorption changes, atebrin and 8-anilinonaphthalene-1-sulphonate fluorescence responses, and phenyldicarbaundecaborane transport.The data obtained are consistent with the suggestion that carotenoid, bacteriochlorophyll and phenyldicarbaundecaborane responses are indicators of Δψ, while an atebrin response is an indicator of ΔpH. The fluorescence of 8-anilinonaphthalene-1-sulphonate is affected both by Δψ and ΔpH.     

Activation-deactivation reactions in the ATPase enzyme in Rhodospirillum rubrum chromatophores

(1) The ATPase enzyme in untreated chromatophores from Rhodospirillum rubrum is in a low-activity state (designated as E°). It can be activated by application of a transmembrane generated by light-induced electron transport, or by application of acid-base jumps. (2) After rapid dissipation of the light-induced , the active state of the ATPase enzyme decays (in the absence of added substrates or products) to a low-activity state (designated as E′), with a half-time of the order of 2–4 min. This state differs from E° in that E′ (but not E°) can be rapidly reactivated by addition of substrate, but only when the Mg²⁺ concentration is kept below 20–30 μM. Since this is characteristic of an activated enzyme containing tightly bound ADP (Slooten, L. and Nuyten, A. (1981) Biochim. Biophys. Acta 638, 313–326), it is suggested that release of endogenous, tightly bound ADP is one of the factors involved in activation of the ATPase enzyme.     

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg²⁺). I₅₀ values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min^?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg²⁺ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg²⁺ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg²⁺. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg²⁺ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg²⁺ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.     

Light-induced oxygen uptake by chromatophores and subchromatophore pigment-protein complexes of Rhodospirillum rubrum]

Chromatophores of R. rubrum incubated with electron donors, e. g. reduced diaminodurene, TMPD, phenazine methosulphate, cytochrome c or ferrocyanide, are able to catalyze O2 uptake upon illumination. This process is inhibited by o-phenanthroline as well as upon extraction of quinones from chromatophores, but not by antimycin A, rotenone or CN-. The O2 uptake sensitive to the action of o-phenanthroline is also observed in the illuminated subchromatophore P870 reaction center complexes and reaction center plus light-harvesting antenna complexes incubated with electron donors, quinones and detergents. The data obtained are in agreement with a suggestion that the photooxidase activity of chromatophores and subchromatophore pigment-protein complexes is due to the interaction of photoreduced ubiquinone with O2.     

Synthesis of free ATP from membrane-bound ATP in chromatophores of Rhodospirillum rubrum

Chromatophores of Rhodospirillum rubrum were preincubated with ³²P_i in the absence of added nucleotides. Particles and reaction mixture were then separated by sucrose density gradient centrifugation. The labeled chromatophores thus obtained esterify ³²P_i into acid-soluble ATP (ATP_as) on the addition of ADP in the dark. Additional firmly bound ATP (ATP_fb) can be liberated on sodium dodecylsulfate treatment. Coinciding with the formation of acid-soluble ATP there is a decrease in the amount of firmly bound ATP. The isotopic concentration experiments in which labeled chromatophores were incubated with carrier-free ³²P_i and ADP in the dark, show that ATP_as might arise from ATP_fb not by a direct γ-phosphate transfer but by an esterification of the added ADP and free phosphate with a concomitant hydrolysis of the ATP_fb. On this basis we have proposed a new working hypothesis for the last step of electron transport-linked phosphorylations. It includes the following reactions: + P*_i → P* (i.e., ATP_fb) P* + ADP + P**_i → ATP**_as + P*_i

The hypothesis is compatible with the concept of conformational energy conservation.     

Reconstitution of electrogenic function in isolated pigment-protein complexes of Anabaena variabilis

Treatment of Anabaena variabilis membranes with lauryldimethylamine N-oxide yielded two fractions of pigment-protein complexes which were separable by gel filtration on Sepharose 6B. A green fraction was characterized which had a maximum of the chlorophyll long-wave absorption band at 678 nm and a small amount of carotenoid. In this fraction, Photosystem I activity was higher than in another (brownish-green) fraction which had a maximum of the chlorophyll absorption band at 673 nm and which was enriched in carotenoids. Similarly to isolated membranes, proteoliposomes containing pigment-protein complexes took up tetraphenylborate anions and tetraphenylphosphonium cations and were found to be capable of light-dependent membrane potential generation, when associated with a planar phospholipid membrane in the presence of reduced phenazine methosulfate upon illumination. The spatial arrangement of the pigment-protein complexes in the native and artificial membranes is discussed.     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

Shifts of the bacteriochlorophyll absorption band at 880 nm in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum]

The redox potential dependency of the light-induced absorption changes of bacteriochlorophyll in the chromatophores and subchromatophore particles from Rhodospirillum rubrum has been studied. The highest values of the absorption changes due to the bleaching of P870 and the blue shift of P800 are observed within the redox potential range of 360--410. At the potential values below 300 mV the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in the subchromatophore particles and to longer wavelengths in the chromatophores. Redox titration revealed that the red and blue shifts of 880 nm bacteriochlorophyll band are caused by the functioning of a non-identified component (X) which has an oxidation -- reduction midpoint potential close to 340 mV (n = 1) within the pH range of 6,0--7,6. The Em for this component decreases by 60 mV/pH unit within the pH range of 7.6--9,2. The results obtained suggest that the red shift is due to the transmembrane, while the blue shift -- to the local intramembrane electric field. The generation of both the transmembrane and local intramembrane electric fields apparently depends on redox transitions of the component X.     

Extraction of chromatophores from Rhodospirillum rubrum in aqueous two-phase systems: A method for large-scale isolation of membrane particles

Chromatophores, membrane vesicles with the capacity of cyclic photophosphorylation, have been isolated from Rhodospirillum rubrum cells on a pilot plant scale. Results of disintegration in a glass bead mill and in a high pressure homogenizer were compared. The chromatophores were isolated from the crude extract by extraction in aqueous two-phase systems. In systems of polyethylene glycol (PEG) and dextran the chromatophores were partitioned to the upper PEG phase by the addition of PEG-palmitate. Most of the proteins and nucleic acids were forced to the bottom phase by addition of sodium chloride. Methods to prevent precipitation of the chromatophores were studied.     

Light- and chemically-induced oxidation-reduction reactions in chromatophore fractions of Rhodospirillum rubrum

Chromatophore fractions of Rhodospirillum rubrum were prepared by centrifugation of a ‘classical’ chromatophore preparation in a sucrose density gradient. Most of the experiments were carried out with a fraction separating out at about 23% sucrose. Although no light-induced reactions of cytochromes could be detected, the presence of two bound cytochromes, C422 and C428, in this fraction was indicated by oxidation-reduction reactions induced by KIO₄ and dithionite. Cytochrome C422 appeared to be the ‘high-potential’ cytochrome detected earlier in preparations from this organism. Titration of chemically-induced absorbance changes due to a reduction of oxidized cytochrome C428 has led to an estimated redox midpoint potential of E_m7.5 = −0.01 V for this component in a four-electron transport reaction.

Light-induced absorbance changes in the near-infrared spectral region measured in the light chromatophore fraction at different environmental redox potentials suggested a bacteriochlorophyll component, P', with spectral characteristics which were different from those of the high-potential bacteriochlorophyll component P890. The spectral shifts observed upon oxidation, either by light or by air were a bleaching in the 860–880-nm spectral region and an appearance of an absorption band at about 900 nm. The component could be oxidized and reduced reversibly and had an estimated midpoint potential of +0.06 V in a one-electron reaction.     

Kinetics of the H-ATPase in chromatophores from Rhodospirillum rubrum: Effect of the electrical transmembrane potential on the rate constants

The effect of the electrical potential on the H⁺-ATPase of Rhodospirillum rubrum is examined. It is shown that the forward reaction rate (ATP synthesis) is increased by a factor of 10 during illumination while the reversed rate is only slightly decreased. This indicates that the electrical potential across the membrane affects the rate constants mainly by increasing the forward rate constants rather than decreasing the reversed rate constants in order to go from net hydrolysis to net synthesis.     

Ubiquinone reduction and proton uptake by chromatophores of Rhodopseudomonas sphaeroides R-26. Periodicity of two in consecutive light flashes

Chromatophores of Rhodopseudomonas sphaeroides strain R-26 were subjected to a series of brief flashes of light in the presence of diaminodurene as an electron donor. Odd-numbered flashes induced the reduction of ubiquinone to the anionic semiquinone, as indicated by absorbance changes near 450 nm. This reaction was not attended by proton binding. Even-numbered flashes caused disappearance of the semiquinone, presumably by conversion to the fully reduced form. This reaction was attended by proton uptake.     

Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360–410 mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores.

The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C₃₄₀) which has an oxidation-reduction midpoint potential close to 340 mV (n = 1) at pH 6.0–7.6. The E_m of this component varies by 60 mV/pH unit between pH 7.6 and 9.2.

The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C₃₄₀.