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1.
The mechanism of light-induced O 2 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 O 2 uptake by the extracted chromatophores and stimulated the O 2 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 O 2. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O 2 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 (Me 4Ph(NH 2) 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 O 2, 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 Me 4Ph(NH 2) 2 to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me 4Ph(NH 2) 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 Me 4Ph(NH 2) 2 and TMPD are different. 相似文献
2.
N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1). 相似文献
3.
N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit steady-state proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. In chromatophores of the purple bacterium Rhodobacter sphaeroides, this has been associated with the specific labeling of a surface-exposed aspartate-187 of the cytochrome b subunit of the bc(1) complex [Wang et al. (1998) Arch. Biochem. Biophys. 352, 193-198]. To explore the possible role of this amino acid residue in the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and the electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores from wild type (WT) and mutant cells, in which aspartate-187 of cytochrome b (Asp(B187)) has been changed to asparagine (mutant B187 DN). The kinetics and amplitude of phase III of the electrochromic shift of carotenoids, reflecting electrogenic reactions in the bc(1) complex, and of the redox changes of cytochromes and reaction center, were similar (+/- 15%) in both WT and B187DN chromatophores. DCCD effectively inhibited phase III of the carotenoid bandshift in both B187DN and WT chromatophores. The dependence of the kinetics and amplitude of phase III of the electrochromic shift on DCCD concentration was identical in WT and B187DN chromatophores, indicating that covalent modification of Asp(B187) is not specifically responsible for the effect of DCCD-induced effects of cytochrome bc(1) complex. Furthermore, no evidence for differential inhibition of electrogenesis and electron transport was found in either strain. We conclude that Asp(B187) plays no crucial role in the protonogenic reactions of bc(1) complex, since its replacement by asparagine does not lead to any significant effects on either the electrogenic reactions of bc(1) complex, as revealed by phase III of the electrochromic shift of carotenoids, or sensitivity of turnover to DCCD. 相似文献
4.
The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-. 相似文献
5.
1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation. 相似文献
6.
N,N′-dicyclohexylcarbodiimide (DCCD) was found to uncouple phosphorylation from oxidation with succinate and NAD +-linked substrates in the system from Mycobacterium phlei. However, in contrast to the effect of this agent in mammalian mitochondria, DCCD was found to stimulate oxidation with succinate as an electron donor and to inhibit the oxidation of NAD +-linked substrates. Furthermore, in the M. phlei system DCCD was found to inhibit the membrane bound latent ATP-ase but had no effect on this activity when the latent ATPase was removed from the membrane vesicles. Reconstitution with the fraction containing latent ATPase activity and the membrane vesicles resulted in inhibition of latent ATPase by DCCD. Studies of the effect of DCCD on the resolved system indicated that DCCD may be associated with membrane vesicles or causes secondary changes in conformation of membrane vesicles. Although DCCD inhibited membrane bound ATPase it did not prevent the addition of the solubilized ATPase to the membrane vesicles. DCCD was found to have no effect on purified succinic dehydrogenase activity but stimulated this activity in the electron transport particles. 相似文献
7.
The effects of isooctane-extraction on the quantum yield ofphotooxidation of cytochromes in chromatophores of Chromatiumvinosum, strain D, were investigated. The initial rate of photooxidation of cytochrome c-555 in theisooctane-extracted chromatophores was decreased by repeatedor prolonged preillumination in the presence of 30 mM ascorbate.The minimum number of light quanta absorbed during preilluminationto cause the maximum decrease in the photooxidation of cytochrome c-555 was about 2% of the number of bacteriochlorophyll moleculespresent. In the absence of ascorbate no lowering of the initial rateof cytochrome photooxidation was observed after prolonged orrepeated illumination. No decrease in the initial rate due topreillumination was observed in lyophilized or ubiquinone-readdedchromatophores. The initial rate of photooxidation of both the cytochromes c-555and c-552 in partially isooctane-extracted chromatophores (5090%extraction of ubiquinone) was also decreased by repeated orprolonged illumination in the presence of 30 mM ascorbate. Our previous and present studies indicate that about 10% ofthe total ubiquinone- 7 functions as the primary electron acceptorfor the photooxidation of cytochrome c-552, and that the majorpart of the ubiquinone functions as the common secondary electronacceptor for the photooxidation of cytochromes c-555 and c-552in Chromalium chromatophores. Therefore, ubiquinone probablyhas dual roles in the light-induced electron transfer of Chromatiumchromatophores. (Received July 23, 1975; ) 相似文献
8.
The effect of high intensity (photosynthesis-saturating) light on the optical properties of the bacteriochlorophyll and the light-induced H+ uptake by R. rubrum chromatophores was studied. It was shown that under aerobic conditions illumination causes reversible inhibition (in the dark) of the chromatophore ability for the light-induced uptake of H+, a reversible inhibition of the photosynthetical reaction center function and irreversible bleaching of the antennal bacteriochlorophyll. A kinetic comparison of spectral effects and reversible changes in pH as well as the effects of atmospheric oxygen and exogenous electron donors suggests that inhibition of photoactivity of the chromatophores upon illumination is due to accumulation of oxidized bacteriochlorophyll in the reaction center. 相似文献
9.
Antimycin A causes a biphasic suppression of the light-induced membrane potential generation in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores incubated anerobically. The first phase is observed at low antibiotic concentrations and is apparently due to its action as a cyclic electron transfer inhibitor. The second phase is manifested at concentrations which are greater than 1--2 muM and is due to uncoupling that may be connected with an antibiotic-induced dissipation of the electrochemical H+ gradient across the chromatophore membrane. The inhibitory effect of antimycin added at low concentrations under aerobic conditions is removed by succinate to a large extent. It is expected that the electrogenic cyclic redox chain in the bacterial chromatophores incubed under conditions of continuous illumination may function at two regimes: (1) as a complete chain involving all the redox components, and (2) as a shortened chain involving only the P-870 photoreaction center, ubiquinone and cytochrome c2. 相似文献
10.
Light-induced proton uptake, light-induced carotenoid absorbance shift, photophosphorylation, and hydrolysis of Mg-ATP, Ca-ATP, and PPi in Rhodospirillum rubrum chromatophores are shown to be inhibited by the antibiotic equisetin. The Mg- and Ca-ATPase activities of purified F0F1-ATPase are inhibited by equisetin. In contrast, only the Ca-ATPase activity of purified F1-ATPase is decreased by equisetin, whereas the Mg-ATPase is stimulated. Both equisetin and N,N'-dicyclohexylcarbodiimide (DCCD) inhibit the hydrolytic activity of the purified H+-PPase but not the hydrolytic activity of soluble PPase from R. rubrum and yeast. The I50 for the PPi hydrolysis is near 20 microM for both equisetin and DCCD. The action of equisetin on membranes is compared to the effect of Triton X-100 and carbonyl cyanide p-trifluoromethoxyhydrazone. On the basis of these new data, equisetin is proposed to act nonspecifically on membranes and hydrophobic domains of proteins. 相似文献
11.
Absorbance changes at 450 nm of the semiquinone form of the secondary electron acceptor were studied in chromatophores of Rhodospirillum rubrum. When chromatophores are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes at ambient redox potential from 100 to 250 mV. A simple kinetic model of the binary oscillations is suggested. On the base of the model it is shown that the rate constant of electron transfer from primary to secondary quinone after the first flash is larger that after the second flash. Cooperativity in electron transfer from primary to secondary quinone can be explained by electrostatic interactions of charged carriers. 相似文献
12.
The light-induced interaction of phenazine methosulfate (PMS) with chromatophores of the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides was studied, using an ion-specific electrode. Illumination caused an initial rapid increase in the concentration of methylphenazinium cation (MP+) and a subsequent slow (1-3 min) decrease of the MP+ concentration to a low steady level. The rapid phase of the light-induced MP+ concentration change is specifically enhanced by ascorbate. The slow phase (uptake of MP+ from the medium) is stimulated on addition of valinomycin, which is known to collapse the membrane potential of energized chromatophores, and is partly inhibited by NH4Cl, which enhances the membrane potential in chromatophores. The light-induced uptake of MP+ is sharply stimulated by dibromothymoquinone. It is concluded that the initial rapid increase of the MP+ concentration in the outer medium results from the oxidation of the reduced PMS by photooxidized reaction centers. The slow decrease of the external MP+ concentration is due to active transport of MP+ into the internal space of the chromatophores via a mechanism of a chemiosmotic type. The accumulation of MP+ is directly mediated by the redox reactions of PMS at the outer and inner surfaces of the photosynthetic membrane, which are involved in cyclic electron transport. 相似文献
13.
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. 相似文献
14.
Radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD) was bound selectively in a time- and concentration-dependent manner to cytochrome b6 of an enzymatically active cytochrome bf complex isolated from spinach chloroplasts. Maximum labeling of cytochrome b6 was observed with 30 nmol DCCD per nmol cytochrome b6 in the cytochrome bf complex incubated for 30-60 min at 12 degrees C. After incubation of the cytochrome bf complex with DCCD under these conditions, the rate of proton ejection in the complex reconstituted into liposomes was decreased approximately 65-70% when compared to controls; however, under these same conditions the rate of electron transfer through either the soluble bf complex or the complex reconstituted into liposomes was only decreased around 20%. These results suggest that the mechanism of proton translocation through the cytochrome bf complex of spinach chloroplasts is similar to that of the cytochrome bc1 complex from yeast mitochondria in which proton pumping but not electron transfer is also inhibited by DCCD (D. S. Beattie and A. Villalobo, 1982, J. Biol. Chem. 257, 14,745-14,752). 相似文献
15.
The electrochemical proton gradient formed during light-induced electron transport in bacterial chromatophores is composed of both a proton concentration gradient and a membrane potential that can interchange under appropriate conditions. Both components, whether light-induced or imposed artificially in the dark, can drive ATP synthesis. 相似文献
16.
N,N'-Dicyclohexylcarbodiimide (DCCD) inhibited dark re-reduction of cytochrome c2 and reduction of b-type cytochrome, both of which are closely associated with electron transfer involving a cytochrome b-c2 oxidoreductase, after a single-turnover flash excitation in the chromatophore membranes from a photosynthetic bacterium, Rhodopseudomonas sphaeroides. Rapid proton uptakes (HI+, HII+) and the formation of the membrane potential registered by carotenoid bandshift phase III were also inhibited by DCCD. The electron transfer was inhibited in the presence of either valinomycin or carbonylcyanide-m-chlorophenylhydrazone (CCCP). These results indicated that DCCD inhibited the electron transfer involving the cytochrome b-c2 oxidoreductase in the bacterium. The inhibition was irreversible. A hydrophilic carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), did not affect the above-mentioned reactions. Thus, DCCD may interact with the hydrophobic region(s) in the chromatophore membranes from photosynthetic bacteria resulting in the inhibition(s) of the photosynthetic cyclic electron transfer. 相似文献
17.
Semiconductor nanocrystals, often known as quantum dots, have been used extensively for a wide range of applications in bioimaging and biosensing. In this article, we report that the pH-sensitive cadmium telluride (CdTe) quantum dots (QDs) were used as a proton sensor to detect proton flux that was driven by ATP synthesis in chromatophores. To confirm that these QD-labeled chromatophores were responding to proton flux pumping driven by ATP synthesis, N,N'-dicyclohexylcarbodiimide (DCCD) was used as an inhibitor of ATPase activity. Furthermore, we applied the QD-labeled chromatophores as a virus detector to detect the H9 avian influenza virus based on antibody-antigen reaction. The results showed that this QD virus detector could be a new virus-detecting device. 相似文献
18.
The carboxyl group reagents dicyclohexylcarbodiimide (DCCD) and N-ethoxycarboxyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivate the soluble Rhodospirillum rubrum F1-ATPase (RrF1). The inactivation is both time- and concentration-dependent and also pH-dependent, being more marked at acid pH. Under the same conditions, N-ethyl-5-phenylisoxazolium 3'-sulfonate causes almost no inactivation of the RrF1-ATPase. Complete inhibition of the enzyme activity requires the binding of 1 mol of DCCD/mol of RrF1. The isolated, reconstitutively active, beta-subunit of RrF1 is affected by the three carboxyl group reagents in a very similar manner to the RrF1-ATPase. Incubation of the beta-subunit with DCCD and EEDQ eliminates its capacity to rebind to beta-less chromatophores. Consequently the DCCD or EEDQ-modified beta-subunit cannot restore ATP synthesis or hydrolysis activities to the beta-less chromatophores. The interaction of the isolated beta-subunit with DCCD and EEDQ is both time and concentration dependent. The elimination of the reconstitutive activity of the beta-subunit by DCCD is accompanied with a covalent binding of about 1 mol of [14C]DCCD/mol of beta and is pH-dependent, showing a half-maximal effect at about pH 7.4. Divalent cations, inorganic phosphate, and to a lesser extent ATP and ADP decrease the binding stoichiometry of DCCD to the beta-subunit. Pretreatment of either RrF1 or its isolated beta-subunit with EEDQ reduces drastically their ability to bind [14C]DCCD, suggesting that in both RrF1 and the beta-subunit, EEDQ and DCCD might react at the same site. The similar effect of the carboxyl group reagents on RrF1 and on its isolated beta-subunit is in accord with the suggestion that DCCD and EEDQ affect the F1-ATPases by interacting with their beta-subunits. 相似文献
19.
The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O 2. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K 4Fe(CN) 6, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c2 from the heavy chromatophores caused a decrease in the light-induced O 2 uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c2 from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c2 level which faces the outside medium. However, cytochrome c2 is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c2 in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O 2 whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O 2 consumed is 4, which means that the end product of O 2 reduction is H 2O. 相似文献
20.
Antimycin A causes a biphasic suppression of the light-induced membrane potential generation in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides chromatophores incubated anaerobically. The first phase is observed at low antibiotic concentrations and is apparently due to its action as a cyclic electron transfer inhibitor. The second phase is manifested at concentrations which are greater than 1–2 μM and is due to uncoupling that may be connected with an antibiotic-induced dissipation of the electrochemical H + gradient across the chromatophore membrane. The inhibitory effect of anti-mycin added at low concentrations under aerobic conditions is removed by succinate to a large extent. It is expected that the electrogenic cyclic redox chain in the bacterial chromatophores incubated under conditions of continuous illumination may function at two regimes: (1) as a complete chain involving all the redox components, and (2) as a shortened chain involving only the P-870 photoreaction center, ubiquinone and cytochrome c2. 相似文献
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