Article

In situ characterisation of photosynthetic electron transport in Rhodopseudomonas capsulata

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Abstract

1. The effects of varying the ambient oxidation/reduction potential on the redox changes of cytochromes c, cytochromes b and P605 induced by a laser flash in chromatophores from Rhodopseudomonas capsulata Ala Pho+ have been investigated.2. The appearance and attenuation of the changes with varying ambient redox potential show that, of the cytochromes present, cytochromes c with Em7 = 340 mV and 0 mV, and cytochrome b, Em7 = 60 mV were concerned with photosynthetic electron flow.3. The site of action of antimycin was shown to be between cytochrome b60 and a component, as yet unidentified, called Z.4. The appearance or attenuation of laser-induced changes of cytochromes c0 and b60 on redox titration was dependent on pH, but no effect of pH on the cytochrome c340 titration was observed.5. The dependence on ambient redox potential of the laser-induced bleaching at 605 nm enabled identification of the mid-point potentials of the primary electron donor (Em7 = 440 mV) and acceptor (Em7 = −25 mV).6. The interrelationship of these electron carriers is discussed with respect to the pathway of cyclic electron flow.

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... The involvement of ubiquinone in the photosynthetic electron-transfer chain of Rhodopseudomonas sphaeroides and Rps. capsulata has previously been discussed in terms of a number of specialized quinones identified by the characteristics of particular reactions of the chain, and the behavior with respect to extraction, redox potential and kinetics of interaction with reaction partners [1][2][3][4][5][6]. ...
... In previous versions of the Q-cycle proposed to operate in Rps. sphaeroides, a central role has been assigned to a special quinone, Q Z , assumed to be present at a specific binding site with a stoichiometry of 0.7 Q Z /reaction center [4,5,7,20]. The redox midpoint potential of Q Z (E m,7 for the couple Q Z /Q Z H 2 of approx. ...
... The redox midpoint potential of Q Z (E m,7 for the couple Q Z /Q Z H 2 of approx. 150 mV, varying by −60 mV/pH unit, n value of 2), has not been measured directly, but has been estimated from the dependence on ambient redox potential (E h ), and pH, of the rate of the antimycin-sensitive reduction of cytochrome c (c 1 and c 2 ) [4,5,20,25]. At values of E h above the supposed E m of Q Z /Q Z H 2 , no rapid reduction was seen; below the E m , cytochrome c was reduced in a reaction of t 1/2 ≈ 1-2 ms which was antimycin sensitive. ...
Article
(1) The role of the ubiquinone pool in the reactions of the cyclic electron-transfer chain has been investigated by observing the effects of reduction of the ubiquinone pool on the kinetics and extent of the cytochrome and electrochromic carotenoid absorbance changes following flash illumination. (2) In the presence of antimycin, flash-induced reduction of cytochrome b-561 is dependent on a coupled oxidation of ubiquinol. The ubiquinol oxidase site of the ubiquinol:cytochrome c(2) oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c(1) and c(2), the Rieske-type iron-sulfur center, and the reaction center primary donor, and a second electron is transferred to a low-potential chain containing cytochromes b-566 and b-561. (3) The rate of reduction of cytochrome b-561 in the presence of antimycin has been shown to reflect the rate of turnover of the ubiquinol oxidase site. This diagnostic feature has been used to measure the dependence of the kinetics of the site on the ubiquinol concentration. Over a limited range of concentration (0-3 mol ubiquinol/mol cytochrome b-561), the kinetics showed a second-order process, first order with respect to ubiquinol from the pool. At higher ubiquinol concentrations, other processes became rate determining, so that above approx. 25 mol ubiquinol/mol cytochrome b-561, no further increase in rate was seen. (4) The kinetics and extents of cytochrome b-561 reduction following a flash in the presence of antimycin, and of the antimycin-sensitive reduction of cytochrome c(1) and c(2), and the slow phase of the carotenoid change, have been measured as a function of redox potential over a wide range. The initial rate for all these processes increased on reduction of the suspension over the range between 180 and 100 mV (pH 7). The increase in rate occurred as the concentration of ubiquinol in the pool increased on reduction, and could be accounted for in terms of the increased rate of ubiquinol oxidation. It is not necessary to postulate the presence of a tightly bound quinone at this site with altered redox properties, as has been previously assumed. (5) The antimycin-sensitive reactions reflect the turnover of a second catalytic site of the complex, at which cytochrome b-561 is oxidized in an electrogenic reaction. We propose that ubiquinone is reduced at this site with a mechanism similar to that of the two-electron gate of the reaction center. We suggest that antimycin binds at this site, and displaces the quinone species so that all reactions at the site are inhibited. (6) In coupled chromatophores, the turnover of the ubiquinone reductase site can be measured by the antimycin-sensitive slow phase of the electrochromic carotenoid change. At redox potentials higher than 180 mV, where the pool is completely oxidized, the maximal extent of the slow phase is half that at 140 mV, where the pool contains approx. 1 mol ubiquinone/mol cytochrome b-561 before the flash. At both potentials, cytochrome b-561 became completely reduced following one flash in the presence of antimycin. The results are interpreted as showing that at potentials higher than 180 mV, ubiquinol stoichiometric with cytochrome b-561 reaches the complex from the reaction center. The increased extent of the carotenoid change, when one extra ubiquinol is available in the pool, is interpreted as showing that the ubiquinol oxidase site turns over twice, and the ubiquinone reductase sites turns over once, for a complete turnover of the ubiquinol:cytochrome c(2) oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c(1) and c(2) is shown to reflect the second turnover of the ubiquinol oxidase site. (8) We suggest that, in the presence of antimycin, the ubiquinol oxidase site reaches a quasi equilibrium with ubiquinol from the pool and the high- and low-potential chains, and that the equilibrium constant of the reaction catalysed constrains the site to the single turnover under most conditions. (9) The results are discussed in the context of a detailed mechanism. The modified Q-cycle proposed is described by physicochemical parameters which account well for the results reported.
... In chromatophores from the purple non-sulfur bacteria, light-induced absorbance changes measured at single or dual wavelengths have been attributed to cytochrome c2 (Em(7.o) 295 mV in Rhodopseudomonas sphaeroides [1,2], Em(v.0) 345 mV in Rhodopseudomonas capsulata [3]), the photochemical reaction center bacteriochlorophyll dimer ((BChl)2) (Em(v.0) 440 mV in Rps. ...
... sphaeroides [1] and Rps. capsulata [3]), cytochrome bs0 (Em(v.0) 50 mV [1,3--5]), cytochrome blss (Em(v.0) ...
Article
1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λmax at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (λmax at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b50 appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b−90 was observed.4. Difference spectra attributed to (BChl)2, Q⨪II, c type cytochrome and cytochrome b50 were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.
... Eine weitere bekannte Funktion von [Kennedy et al., 1983; Emptage et al., 1983). Ebenfalls erwähnenswert sind hierbei kleine High-Potential-Iron-Sulfur-Proteine (6-10 kDa), deren relativ hohes Redoxpotential von 0 bis +500 mV (Dus et al., 1967) Elektronenübertragungsprozesse in der Photosynthese (Evans & Crofts, 1974) bzw ...
... The rate of ferricytochrome cz reduction is stimulated at least lo-fold when the 2 ubiquinone is reduced before activation. 6,9,25,26 2. The third phase of the carotenoid bandshift, which is presumed to monitor a transmembrane electrogenic event in the 6, 21 region of cytochrome b, occurs if the 2 ubiquinone is reduced prior to activation. 3. The rate of cytochrome b reduction is stimulated at least 5-fold when the Z ubiquinone is reduced before activation. ...
Article
Full-text available
Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.
... In the organisms examined, one or more of the reaction center subunits (H, M, or L) are exposed at the periplasmic surface of the membrane. During cyclic photophosphorylation soluble and membrane c-type cytochromes (or multiple membrane-associated c-type cytochromes) have been shown to reduce the reaction center (47, 48, 63,68,113,172,188,200,233,236). The soluble cytochrome (cytochrome c2) has been localized in the periplasm of Rhodopseudomonas sphaeroides and R. capsulata (187). ...
... To avoid fast, unresolved re-reduction of P + by cyt c 2 following a flash of light, measurements were performed at an ambient redox potential of E h = 420 mV, at which the main endogenous electron donor to P + , cyt c 2 (E m = 350 mV [47]), is completely pre-oxidized in the dark. For the P + /P couple, a E m value of 440 mV was assumed [48] for correcting the fraction of the primary donor pre-oxidized at the redox poise of the measurement. Fig. 1 and Fig. 7 were generated using the Swiss-PdbViewer software (http:// www.expasy.org/spdbv/) ...
Article
Full-text available
The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn(2+) binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.
... In the organisms examined, one or more of the reaction center subunits (H, M, or L) are exposed at the periplasmic surface of the membrane. During cyclic photophosphorylation soluble and membrane c-type cytochromes (or multiple membrane-associated c-type cytochromes) have been shown to reduce the reaction center (47, 48, 63, 68,113,172,188,200,233,236). The soluble cytochrome (cytochrome c2) has been localized in the periplasm of Rhodopseudomonas sphaeroides and R. capsulata (187). ...
... The oxidized minus reduced spectrum of the primary donor of Rhodoferax reaction centre resembles that of other purple bacteria [14,24,25]. The midpoint potential of the primary acceptor (13 mV) is similar to that measured in species containing ubiquinone as QA (e.g., Rhodobacter sphaeroides (-20 mV) [13,32] and Rhodobacter capsulatus (-25, -30 mV) [33,34]). Since a large amount of ubiquinone was found in Rhodoferax membranes [18,19], we suggest that also in this species ubiquinone acts as the primary (QA) and secondary (Qs) acceptor. ...
Article
Flash-induced oxidation of the membrane-bound c-type haems was studied in light-grown cells of Rhodoferax fermentans, a new taxon of the purple nonsulfur photosynthetic bacteria. At least three c-type cytochromes were found to rapidly (< 20 mu s) re-reduce the photo-oxidized primary donor of the reaction centre: a first haem peaking at 556 nm with E(m) = 354 mV, a second haem peaking at 560 nm with Em = 294 mV, and a third haem peaking at 551 nm with E(m) = 79 mV. The photo-oxidized minus reduced spectrum of the primary donor was found to be very similar to those of other purple bacteria. The primary donor midpoint potential was determined (471 mV) and o-phenanthroline was found to inhibit electron transfer to the secondary acceptor, thus indicating the presence of a Q-type reaction centre. The midpoint potential of the primary quinone acceptor was also determined (13 mV). A model accounting for the post-flash redox equilibria within the RC-cytochromes c chain is presented. A tetrahaem cytochrome c is proposed to operate in Rhodoferax fermentans and its spectral and thermodynamic features are discussed in relation with other species of purple photosynthetic bacteria.
... In fact, the initial step of PIET has been confirmed for various donor-acceptor pairs doped in solid polymer films to be significantly influenced by an electric field [24][25][26][27][28][29], based on the measurements of the electric-field effects on fluorescence whose process competes with the PIET. In the realm of nature, e.g., in photosynthetic reaction center, it has been also pointed out that the initial step of PIET is influ- enced by an electric field produced by protein membranes [30][31][32][33][34]. ...
Article
External electric-field-induced change in fluorescence spectra as well as in fluorescence decay has been measured for electron donor and acceptor pairs of pyrene (PY) and N-methylphthalimide (NMPI) doped in a polymer film. Field-induced quenching and field-induced shortening of lifetime are observed for fluorescence emitted from the locally excited (LE) state of PY, indicating that intermolecular electron transfer from the excited state of PY to NMPI is enhanced by an electric field in a polymer film. A simulation has been made for the field effect on decay profile of the LE fluorescence of PY. Exciplex fluorescence is also quenched by an electric field because of the field-induced decrease in the initial population of the fluorescent exciplex. Both in LE fluorescence of PY and in exciplex fluorescence, electric-field-induced quenching becomes less efficient in the presence of a magnetic field. The mechanism of the synergy effect of electric and magnetic fields on fluorescence has been discussed.
Article
The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.
Article
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.
Article
(1) Inhibition of cyclic phosphorylation in chromatophores of Rhodopseudomonas capsulata by antimycin A can be fully reversed by artificial redox mediators, provided the ambient redox potential is maintained around 200 mV. The redox mediator need not be a hydrogen carrier in its reduced form, N-methyl-phenazonium methosulfate and N,N,N',N'-tetramethyl-p-phenylenediamine being equally effective. However, the mediator needs to be lipophilic. Endogenous cyclic phosphorylation is fastest around 130 mV. A shift to 200 mV can also be observed if high concentrations of artificial redox mediator are present in the absence of antimycin. (2) ATPase activity of Rhodopseudomonas capsulata, in the light as well as in the dark, activated or not activated by inorganic phosphate, can also be stimulated by N-methylphenazonium methosulfate. This stimulation is highest at redox potentials between 60 to 80 mV and is sensitive to antimycin A. In this case N,N,N',N-tetramethyl-p-phenylenediamine is much less effective.
Article
Dibromothymoquinone has been shown to inhibit light-induced cytochrome b reduction, and oxidation of succinate and NADH by chromatophores of Rhodopseudomonas capsulata. The half-inhibitory concentration of light-induced reactions and NADH oxidation is 2.5 muM, but of succinate oxidation is 16 muM. Hexane extraction inhibited oxidation of NADH and succinate equally. The results are interpreted to suggest that ubiquinone is concerned in all three processes described, but that the pools associated with NADH and succinate oxidation are not equally accessible to dibromothymoquinone.
Article
Cytochrome c2 was removed by washing from heavy chromatophores prepared from Rhodopseudomonas capsulata cells. The easy removal of the cytochrome could indicate that it was attached on the outside of the membrane. Therefore, the membrane was probably oriented inside out in relation to the membrane of regular chromatophores, from which cytochrome c2 could not be removed. Washing of the heavy chromatophores caused loss of photphosphorylation activity. The activity was restored to the resolved heavy chromatophores by the supernatant obtained during the washing or by the native cytochrome c2, which was found to be the active component in this supernatant. The activity could not be restored by other c-type cytochromes. Ascorbate, which enhanced photophosphorylation activity in the heavy chromatophores at the optimal concentration of 8 mm, restored this activity to the washed heavy chromatophores, but at an optimum concentration of 50 mm. Cytochrome c2 and dichlorophenol indophenol reduced the optimum of the ascorbate concentration to 7 mm. This might indicate that the effect of ascorbate is mediated through cytochrome c2. Washing the heavy chromatophores caused 70% loss of the light-induced electron transport from ascorbate and from ascorbate-reduced dichlorophenol indophenol to O2. However, this effect was only observed with the lower concentrations of ascorbate and the dye. The activity was restored either by the supernatant obtained from the washing or by various c-type cytochromes, reduced by ascorbate. Washing the heavy chromatophores did not affect succinate oxidation in the dark. It is suggested that cytochrome c2 is one of the cytochromes catalyzing the photosynthetic cyclic electron transport, as has been seen from its high specificity in the reconstitution experiments. Light can induce oxidation of various c-type cytochromes and other redox reagents. However, reduction was specific for cytochrome c2 from Rps. capuslata, since it was the only one which could be both reduced and oxidized as required from a component which is part of a cyclic electron transport chain. It is also suggested that cytochrome c2 was not part of the succinate oxidase system.
Article
Abstract— Investigations in which EPR has been used as a probe of the mechanism of the primary quantum conversion reaction and/or electron transport reactions in bacterial photosynthesis are surveyed. These investigations include studies of whole cell organisms and simpler sub-cellular preparations, chromatophores and bacteriochlorophyll-protein complexes. Electron paramagnetic resonance studies have successfully demonstrated that the primary donor of the photosynthetic, photochemical reaction involves a dimer of bacteriochlorophyll, generally referred to as P870. P870 is photochemically oxidized to a cation radical which exhibits a g= 2.0025 EPR signal. Comparative studies of the time behaviour of this signal in whole cells and in sub-cellular preparations show that electron flow in the whole cells is substantially different than in the cell-free systems. The primary acceptor molecule of the photochemical reaction has not been conclusively identified. When it is photochemically reduced, it exhibits a broad EPR absorbance centered at g= 1.8 observable only at low temperatures. This signal involves an iron atom and a quinone molecule. Two possible identifications of the species responsible for the g= 1.8 signal are an iron-quinone complex and an iron-sulfur protein. The latter identification would require that one primary acceptor function for two primary donors and that the removal of a tightly held quinone alter the integrity of the system so as to inhibit the photochemistry. When the primary acceptor is chemically reduced, a photo-induced, polarized triplet EPR spectrum is detected. Both absorption and emission lines are, observed as if only one substate (m= 0) of the triplet manifold is populated. The zero field parameters of the triplet spectrum suggest that the triplet is formed through a decay of a biradical, not through an optical singlet to triplet transition. Low temperature EPR studies of photosynthetic preparations which had been poised at room temperature by subjecting the preparations to different redox potentials and/or dark adaptation and illumination show the presence of a number of light-influenced, EPR active components. The spectral characteristics of these components are indicative of iron-heme (both low and high-spin forms) and iron-sulfur (both oxidized and reduced) proteins and at least one other organic free radical distinct from P870+. The spectra varied for different strains of bacteria. Also, some of the signals detected in the whole cell organism were not detected in cell free preparations and the kinetics of the light influenced signal amplitudes were different in the whole cells than in chromatophores.
Article
On looking back at a lifetime of research, it is interesting to see, in the light of current progress, how things came to be, and to speculate on how things might be. I am delighted in the context of the Mitchell prize to have that excuse to present this necessarily personal view of developments in areas of my interests. I have focused on the Q-cycle and a few examples showing wider ramifications, since that had been the main interest of the lab in the 20 years since structures became available, − a watershed event in determining our molecular perspective. I have reviewed the evidence for our model for the mechanism of the first electron transfer of the bifurcated reaction at the Qo-site, which I think is compelling. In reviewing progress in understanding the second electron transfer, I have revisited some controversies to justify important conclusions which appear, from the literature, not to have been taken seriously. I hope this does not come over as nitpicking. The conclusions are important to the final section in which I develop an internally consistent mechanism for turnovers of the complex leading to a state similar to that observed in recent rapid-mix/freeze-quench experiments, reported three years ago. The final model is necessarily speculative but is open to test.
Article
1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E'0 at pH 7.0 +413+/-5, +270+/-5, +148+/-5, +56+/-5 and -32+/-5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b148, b56, and b-32) vary as a function of pH with a slope of 30 mV per pH unit. 2. In the presence of a CO/N2 mixture, the apparent E'0 of cytochrome b270 shifts markedly towards higher potentials (+355mV); a similar but less pronounced shift is apparent also for cytochrome b150. The effect of CO on the midpoint potential of cytochrome b270 is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain. 3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c2 and of virtually all cytochrome cc'. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c2 in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c2 nor the CO-binding cytochrome cc' are involved in this pathway. 4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in C0-difference spectra and with an alpha band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b270, can be classified as a typical cytochrome "0" and considered the alternative CO-sensitive oxidase.
Article
Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.
Article
Formation of the photosynthetic apparatus was induced in aerobically grown dark cultures of Rhodopseudomonas capsulata by lowering of the oxygen tension. Besides the wild type strain the carotenoid-less mutant strain A1a+ was investigated. Both strains exhibited initially a decrease of the molar ratio of total bacteriochlorophyll (Bchl) to reaction center (RC) Bchl, followed by an increase. Synthesis of RC-Bchl preceded the synthesis of light-harvesting (LH) Bchl. Activities of photophosphorylation in membrane preparations, isolated from cultures after different periods of incubation at low aeration, decreased on the basis of total Bchl from about 9 to 2 μmole ATP/μmole total Bchl·min, whereas the rate on the basis of RC-Bchl remained constant (about 500 μmole ATP/μmole RC-Bchl·min). Under the same conditions the membrane proteins were labelled with U−14C-protein hydrolysate. Corresponding to RC-Bchl the synthesis of RC-proteins dominated during the first 30 min of incubation at pO2 below 3 mmHg. After 45–60 min of membrane formation at low aeration the synthesis of LH-complex proteins exceeded the synthesis of RC proteins. The correlations between protein and Bchl synthesis in the sequential formation of RC- and LH-complexes are discussed.
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A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.
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The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c1 and b of the cytochrome bc1 complex, alone or in combination with deletion of the gene for cytochrome c2. Deletion of cytochrome c1 renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c2, because in the absence of the bc1 complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c2 is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc1 complex. The deletion of cytochromes c1 and c2 reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with Em7 = +312 mV and Em6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.
Article
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, , 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.
Article
Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc1 complex perhaps involving a direct QB-Qc site interaction. This latter reaction is suppressed by occupancy of the Qc site, not only by antimycin but by ubiquinol and ubiquinone.
Article
Data on structure and function of the Rieske/cytb complex from Heliobacteria are scarce. They indicate that the complex is related to the b 6f complex in agreement with the phylogenetic position of the organism. It is composed of a diheme cytochrome c, and a Rieske iron–sulfur protein, together with transmembrane cytochrome b 6 and subunit IV. Additional small subunits may be part of the complex. The cofactor content comprises heme c i, first discovered in the Qi binding pocket of b 6f complexes. The redox midpoint potentials are more negative than in b 6f complex in agreement with the lower redox midpoint potentials (by about 150 mV) of its reaction partners, menaquinone, and cytochrome c 553. The enzyme is implicated in cyclic electron transfer around the RCI. Functional studies are favored by the absence of antennae and the simple photosynthetic reaction chain but are hampered by the high oxygen sensitivity of the organism, its chlorophyll, and lipids.
Article
1.1. A proton is involved in the oxidation and reduction of the b-type cytochromes of the photosynthetic membranes of Rhodopseudomonas sphaeroides throughout the physiological range. Of the three cytochromes, b155, b50 and b−90 (the subscripts refer to Em values at pH 7.2), a pK is identified on cytochrome b50. This pK is about pH 7.4 and is most likely to be associated with the reduced form such that it does not involve the proton above the pK but does at pH values below the pK. The pK appears to be functional on a time scale for electron transport such as would be encountered in vivo.2.2. The kinetics of cytochrome b50 reduction are modified by the state of reduction of after the flash at pH values below the pK's of ubiquinone and cytochrome b50. At pH 6 and Eh 220 mV, i.e., P+ reduced immediately by cytochrome c2, the half-time is 1–2 ms; at Eh 380 mV, i.e., P+ sustained after the flash, the half-time is 25–30 ms. At pH 9, the half-time is 8–10 ms, irrespective of whether or not P+ is sustained or immediately reduced after the flash.3.3. The results are discussed in terms of cytochrome b50 and its functional position in the membrane and its interaction with ubiquinone and the handling of protons and in terms of energy conversion processes.
Article
This review focuses on the electrochemical and spectroelectrochemical studies that gave insight into redox potentials of the four mitochondrial complexes and their homologues from bacterial respiratory chains using O2 as a terminal acceptor, thus providing crucial information about their reaction mechanism. Advantages and limitations of the use of the different techniques for the study of membrane proteins are presented. Electrocatalytic experiments are described that revealed specific features of the reaction with the substrates and inhibitors. An overview is given on the great variability of the redox and catalytic properties of the enzymes in different organisms that may be due to adaptation to the specific environments in which these enzymes function. The adaptation of the redox chain to the different types of quinone and substrates is analyzed, and future studies are discussed.
Chapter
This chapter seeks to place our extensive knowledge of the structure and properties of bacterial cytochromes c within the context of the energy-conserving electron transport systems of which they are a part. The role of cytochrome c in mitochondrial electron transport has been considered in detail in Chapter 2 but for comparative purposes in this chapter we will treat both the mitochondrion and the chloroplast as specialised endosymbiotic bacteria. Justification for this derives from the strong similarities between certain aerobic bacteria and mitochondria, on the one hand, and between cyanobacteria and the chloroplast, on the other (Vol. 2, Chap. 6).
Article
Computer simulation of photoexcited electron-donor D* fluorescence quenching in a rigid matrix has been performed. Three processes are assumed to occur: (1) the natural decay of D* excitation, (2) the electron transfer from D* to acceptor A, (3) migration of the excitation energy among D molecules. The effect of an external electric field F on process (2) has been included into considerations. Fluorescence decay curves calculated for various concentrations of D and the field-induced variation in the donor fluorescence intensity have been compared with respective experimental data and the results obtained from the approximate analytical calculations (hopping model).
Article
Photosynthetic bacteria are unique among the eubacteria in their ability to use light as the ultimate energy source (Engelmann, 1888; van Niel, 1932). They are also unique among other photosynthetic organisms in their inability to use water as an ultimate reductant, and consequently they do not evolve oxygen and require the addition of other reductants. Their ability to grow photosynthetically under strictly anaerobic conditions led bacterial systematists to set them off in a special major subgroup.
Article
(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b50 and cytochrome b-90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q{minus sign, dot below}I) and the secondary acceptor ubiquinone (QII) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b50 reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b50 reduction increased about 10-fold as the Eh(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with Em(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100-500 μs before cytochrome b50 reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q{minus sign, dot below}I and QII. It was an effective inhibitor of cytochrome b50 photoreduction at Eh(7.0) 390 mV, but not at Eh(7.0) 100 mV. At the latter Eh, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q{minus sign, dot below}I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At Eh(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b50 reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b50 oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high Eh values. However, at lower redox potentials, cytochrome b50 oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b-90 in the rapid reactions of the cyclic electron transfer chain.
Article
The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Q(pool)) amounts to about 80% of the total ubiquinone and has an E(m7) value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Q(pool)-cyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Q(pool) in different redox states, by the presence of inhibitors specific for the Q(o) site (myxothiazol and stigmaiellin) and Q(i) site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Q(o) site structure. Evidence suggests that the Q(o) site can accommodate two ubiquinone molecules. One (designated Q(os)) is bound relatively strongly and is second only to the ubiquinone of the Q(A) site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Q(o) site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at g(x) at 1.783 (Q) and g(x) at 1.777 (QH2); titration of the line-shape change reveals an E(m7) value of approximately 95 mV. The other molecule (Q(ow)) is bound more weakly, in the same range as the ubiquinone of the Q(B) site of the reaction center. Again, the affinities of the Q form (g(x) at 1.800) and QH2 form (g(x) at 1.777) are nearly equal, and the E(m7) value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Q(o) site double-occupancy model is considered that builds on the previous model based on Q(o) site mutants [Robertson, D. E., Daldal, F., & Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2Fe-2S] cluster ligands of the R. capsulatus cyt bc1 complex are histidines [Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bc1 complex completes a full enzymatic turnover without necessary exchange of ubiquinone with the Q(pool).
Article
This chapter offers information on photosynthetic electron transfer. The overall process of photosynthetic electron transfer is promoted by an array of catalytic proteins, only a few of which are real photochemical enzymes. These proteins form a number of well-defined complexes, partially independent from each other, interacting through redox carriers, freely diffusable either in the membrane lipids or at the membrane-water interface. The concept of membrane photosynthetic complex is experimentally justified by the possibility of isolating specific multiprotein associations, following micellization of the membrane with mild detergents.. In a general sense the complexes present in photosynthetic membranes can be subdivided into photosynthetic reaction centers, usually associated with pigment antenna complexes, and in non-photosynthetic, more conventional, electron transfer complexes. The role of the former is that of photocatalyzing the donation of an electron from a more positive electron donor to a more negative acceptor. Four different types of reaction centers (RC) are known: two photosystems I and II (PSI and PSII) present in the membranes of cyanobacteria and all eukaryotic photosynthetic organisms, and the two types of RC present in purple and green photosynthetic bacteria, respectively.
Article
Photosynthetic electron flow from the cytochrome bc1 complex to the reaction center has been studied in a strain of Rhodopseudomonas capsulata which has had the gene for cytochrome c2 deleted from its genome. Previously, cytochrome c2 was thought to be essential for electron flow between these two complexes, but we find this not to be the case in R. capsulata. Indeed, in this organism it seems likely that cytochrome c1 is able rapidly (t1/2 < 100 μs) to transfer electrons directly to the reaction center. However, this reaction is incomplete; only some 20% of the reaction centers are reduced in this way. In the wild type, a further 20% is rapidly reduced by cytochrome c2, but the remaining reaction centers are reduced rather more slowly by an as yet unidentified route that may involve cytochrome c2 shuttling between complexes. The deletion of the cytochrome c2 gene allows a determination of the oxidation-reduction midpoint potential of cytochrome c1 in the absence of cytochrome c2: cytochrome c1 has an Em7 of 345 mV. Furthermore, the finding that a phase of the electrogenic carotenoid bandshift accompanies the oxidation of cytochrome c1 in the absence of c2 indicates that the heme of cytochrome c1 must be near the inner aqueous-membrane interface of the chromatophore.
Article
Investigations on the bacterial photosynthetic reaction center have recently made several important steps forward. Progress has been made in measuring the time course of the light-driven reactions, and in understanding the thermodynamics of these processes, in determining the chemical-structural properties of the protein and its constituents, and in elucidating the functional relationship of the reaction center with the chromatophore membrane. Although the well-characterized Rhodopseudomonas sphaeroides reaction center has been the main exploratory vehicle in many of these studies, we now have an ever increasing body of information from bacteria of other species and genera. This work is providing information from which we can underline features that are common to bacterial reaction centers, but it also reveals differences which may reflect different selection pressures on the separate species. In this report we shall describe the early photochemical events of the reaction center, summarize the comparative biology of the reaction center, and discuss some of the current physical-chemical problems pertaining to the redox components of the reaction center.
Article
The uptake of permeant tetraphenylborate (TB–) anions and tetraphenylphosphonium (TPP+) cations by the illuminated Rhodospirillum rubrum cells has been studied.Antimycin A causes a biphysic inhibition of the light-induced TB– uptake by the bacterial cells incubated anaerobically. The first phase is observed at low antibiotic concentrations and apparently due to its action as a cyclic electron transfer inhibitor. The second phase is observed at concentrations upwards of 1 M and due to an action of the antibiotic as a photophosphorylation uncoupler. The inhibitory action of antimycin is considerably increased under aerobic conditions; this effect is removed by succinate. It is expected that the intracellular chromatophore cyclic redox chain generating the membrane potential and causing the uptake of the TB– anions under conditions of the 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 c 2.Based on the results of TPP+ uptake by the bacterial cells, a system of the light-induced membrane potential generation in the cytoplasmic membrane is sensitive to antimycin and functions only at a regime dependent on cytochrome b.
Article
The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems “in vivo”.
Article
Membranes from cells of Rhodopseudomonas capsulata grown anaerobically in the dark on glucose plus dimethyl sulfoxide differ from those obtained from photoheterotrophically grown cells in several ways: (a) there are qualitative and quantitative variations in the cytochrome composition; (b) electron-transport rates are unusually low in the cytochrome b to cytochrome c region; (c) light-induced ATP synthesis is dependent on the ability of the alternate respiratory pathway to maintain the Q10-cytochrome b complex in a partially oxidized state; (d) a non-energy-conserving NADH-dehydrogenase activity dominates the respiratory activity. In addition, data obtained with both wild-type and mutant cells that contain altered electron-transport systems tend to exclude a role of the redox chain as ATP-producing machinery during anaerobic/dark growth.
Article
Cells of Rhodopseudomonas capsulata were grown in a turbido-stat and adapted to high (1400 W/m2) or low (40 W/m2) light intensities. In high-light-grown cells the specific BChl content was about 10-times lower, the number of intracytoplasmatic membrane vesicles smaller by a factor of about 20, the photosynthetic unit smaller by a factor of 1.9 and the reaction center content about 5-times lower than in low-light-grown cells. However, the photophosphorylation rate per reaction center under saturating light was higher in high-light-grown cells by a factor of 7.7, apparently compensating the lower amount of reaction centers. Adaptation of the cells to different irradiances not only seems to comprise a variation of the size and composition of the antennae, but also a change in the affinity of the photosynthetic system to light, as concluded from saturation curves obtained from the two adaptation stages of cells.
Article
1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.
Article
Full-text available
The light-driven and the ATP-driven reduction of nicotinamide adenine dinucleotide (NAD) catalyzed by the chromatophore fraction of Rhodopseudomonas capsulata was investigated. Efficient electron donors for the photoreduction of NAD are molecular hydrogen and succinate. In the ATP-dependent reaction system, succinate is a more efficient electron donor than H 2 . The energydependent NAD-reduction is driven by ATP, but not by pyrophosphate or ADP. Oligomycin stimulates the NAD-photoreductions and completely inhibits the ATP-driven NAD-reductions. Rotenone and piericidin A are inhibitors for both the light-driven and the ATP-driven NAD-reductions. Antimycin A is an inhibitor only for the light-driven reductions. The H 2 -linked NAD-photoreduction is less sensitive to these inhibitors and to the uncoupler desaspidin than the succinate-linked reduction. Atebrine, carbonyl cyanide-m-chlorophenylhydrazone, 2,4-dinitrophenol and phenazonium methosulfate are inhibitors for the light-driven and the ATP-driven reductions. Some of the compounds used as inhibitors of the NAD-reduction were also investigated with concerns to their inhibitory effects on cyclic photophosphorylation and O 2 -linked oxidations of reduced NAD, succinate and H 2 . Based on the results of these inhibitor studies, the relationships between cyclic photophosphorylation, light-induced noncyclic electron transport and energy-dependent NAD-reduction are discussed.
Chapter
Absorption spectra measurements are routinely used to assay the cytochromes. Because of the high concentration of strongly absorbing carotenoids and chlorophylls in wild strains of the photosynthetic bacteria, it is difficult to detect cytochrome absorption bands with a hand spectroscope. In addition, the effect of light scattering makes it difficult to measure cytochrome spectra of whole cell suspensions even with many sensitive spectrophotometers. Reduced-minus-oxidized difference spectra between two initially equivalent buffered samples must be resorted to for an indication of the cytochrome content of a suspension of cells or of membrane fragments. With a split-beam spectrophotometer, such as a Cary 14 (or 15) instrument, one of the pair of samples may be left unaltered, chemically oxidized with a small amount of potassium ferricyanide or even sodium hypochlorite, or exposed to photoactive light to test for a light-induced reaction. The other sample may be reduced by endogenous reductants, or by the requisite amount of chemical reductants, such as succinate, ascorbate, NADH, sodium dithionite, or thiol compounds, such as 2-mercaptoethanol or dithiothreitol. Alternatively, a dual wavelength spectrophotometer may be used. The same techniques are applicable to cell-free extracts and purified cytochromes.
Article
This chapter discusses the photochemical reaction centers from Rhodopseudomonas spheroids. The hallmark of the known bacterial photosynthetic reaction centers is the presence of a bacteriochlorophyll (BChl) molecule that is specialized to act as a primary photochemical electron donor. This BChl, called “P870,” “P890,” and so on, after the wavelength of its long-wave absorption maximum, is oxidized by light, while an unspecified electron acceptor becomes reduced. Oxidation of P870 is signaled by loss of the long-wave absorption band (bleaching). In living cells, the oxidized P870 is reduced by one or more c-type cytochromes. In purified reaction centers, the source of electrons for the re-reduction of oxidized P870 depends on the environment. In any case, the defining assay for reaction centers is the reversible light-induced bleaching of P870. In the reaction centers made from Rhodopseudomonas spheroides or Rhodospirillum rubrum, the bleaching of P870 is accompanied by blue shift of a band near 800 nm because of another specialized BChl, P800. There appear to be two or three P800 for each molecule of P870. The absorption increase at 780 nm caused by this blue shift is often easier to measure than the bleaching at 870 nm. Rhodopseudomonas viridis contains the longer-wave pigment BChl b. Reaction centers made from this organism contain P830 and P960, analogous to the P800 and P870 of Rhodopseudomonas spheroides. The blue shift of P830 can be measured as an increase in optical density at 810 nm.
Article
Two carotenoid less mutant strains of Rhodopseudomonas capsulata were isolated. The strain A1a pho- was not able to grow photosynthetically and to synthesize bacteriochlorophyll. However, this organism produced protochlorophyll (phytol ester of Mg-2-vinylpheoporphyrin a5 monomethylester) and protopheophytin. Both pigments were excreted as a macromolecular complex. The intracellular membrane system was poorly developed. A revertant strain of A1a (pho+) was isolated which was able to grow anaerobically in the light as well as aerobically in the dark. The generation time under photosynthetic conditions amounted to 16 hrs whereas under aerobic conditions in the dark that was found to be 2.8 hrs. In addition to bacteriochlorophyll, which was found exclusively in the membrane fraction, protochlorophyll and protopheophytin were synthesized and excreted. A small amount of these pigments was also found intracellulary in the membrane fraction. The structure of the well developed intracytoplasmic membrane reticulum was described.
Article
The light-particle preparations, described previously as derived from chromatophore fractions of Chromatium, exhibit absorbance changes on steady-state illumination with actinic light. Conditions for optimal absorbance changes were established and used to study the effects of variation in redox potential effective at the light-activated electron transport system. The results indicate that at least six components participate in photo-induced changes in oxidation states; two of these can be correlated with the known cytochrome components—cytochrome c−552 and cytochrome c−555. The natures of the remaining components remain to be established. A model is proposed for the electron transport mechanisms activated by light, the salient feature of which is the existence of two functionally different pathways of photo-activated electron movement. One of these involves a pigment-heme protein complex consisting of the 890 mμ active center bacteriochlorophyll coupled to cytochrome c−555, cytochrome cc′ and a third cytochrome (component No. 3); the other consists of a 905 mμ active center bacteriochlorophyll coupled to cytochrome c−552 and a low potential component (P−135). A rationalization of all observations on photometabolism of Chromatium on the basis of this model is presented.
Article
1. Three principal phases of the carotenoid band shift in Rhodopseudomonas spheroides chromatophores elicited by a single-turnover flash can be resolved both kinetically and potentiometrically. Phase I (complete in <1 μs) is apparent over the redox potential limits of the light reaction, i.e. the potential range in which reaction centre bacteriochlorophyll is reduced and the primary electron acceptor oxidized before the flash; thus the band shift is consistent with its response to the formation of P+X−. Phase II (about 25% of the amplitude of Phase I) with an approximate 0.15-ms half time is observed if cytochrome c (Em7.2 + 295 mV) is chemically reduced before the flash and hence may be in response to the photooxidation of cytochrome c and re-reduction of P+. A much slower phase (PHase III) can also be detected at positive potentials. It is enhanced both in extent and formation rate ( to about 1 ms) over the 150 mV potential range in which cytochrome b155 (Em7.2 + 155 mV) becomes chemically reduced. Between 50 and 100 mV this phase is approx. 80–100% of the extent of Phase I. Phase III is abolished by antimycin A as are the oxidation of cytochrome b155 and the re-reduction of photooxidized cytochrome c. All phases are additive. Thus the formation of the carotenoid band shift is in response to pulsed electron transfer events. Further, using multiple, one-turnover flashes, the extent following each flash, and behaviour of the formation the carotenoid band shift can be clearly explained in terms of the electron flow patterns in the chromatophore.
Article
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 Em = +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 1×103. 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.
Article
The light-particle preparations, described previously as derived from chromatophore fractions of Chromatium, exhibit absorbance changes on steady-state illumination with actinic light. Conditions for optimal absorbance changes were established and used to study the effects of variation in redox potential effective at the light-activated electron transport system. The results indicate that at least six components participate in photo-induced changes in oxidation states; two of these can be correlated with the known cytochrome components-cytochrome c-552 and cytochrome c-555. The natures of the remaining components remain to be established. A model is proposed for the electron transport mechanisms activated by light, the salient feature of which is the existence of two functionally different pathways of photo-activated electron movement. One of these involves a pigment-heme protein complex consisting of the 890 mμ active center bacteriochlorophyll coupled to cytochrome c-555, cytochrome cc′ and a third cytochrome (component No. 3); the other consists of a 905 mμ active center bacteriochlorophyll coupled to cytochrome c-552 and a low potential component (P-135). A rationalization of all observations on photometabolism of Chromatium on the basis of this model is presented.
Article
1The b- and c-type cytochromes in chromatophores from photosynthetically grown Rhodospirillum rubrum and Rhodopseudomonas spheroides have been characterized in situ in terms of their oxidation-reduction potential properties.In R. rubrum only one cytochrome c measured at 551—540 nm was resolved potentiometrically; the midpoint potential at pH 7.2, Em7.2, is +293 mV. Three cytochrome species measured at 562—540 nm were resolved; they had Em7.2 values + 170 mV, −5 mV and −105 mV and were tentatively identified with cytochrome b, cc′ and b respectively; they contributed to the over-all absorbance change in the ratio 8:25:17, respectively.In Rps. spheroides the Em7.0 of cytochrome c is +295 mV. Three cytochromes tentatively considered to be of the b-type have Em7.2 values of +155 mV, +50 mV and −90 mV; their approximate reduced minus oxidized maxima were determined to be 558—9 nm, 560 nm and 564 nm, respectively. The approximate relative absorbance ratio given at the reduced minus oxidized maxima was 8:21:14.2The rapid oxidation-reduction reactions of reaction-center bacteriochlorophyll, cytochrome c and cytochromes bin Rps. spheroides measured following a 20-nsec laser flash or a 200-μsec xenon flash have been studied in the absence and presence of antimycin A as a function of oxidation-reduction potential. The results indicated that the reaction-center bacteriochlorophyll (Em7.0+ 450 mV) serves to oxidize the cytochrome c with a midpoint potential of 295 mV, cytochrome c295, (half-time t1/2↗100 μsec with a slow phase t1/2∼2 msec) which in turn can oxidize cytochrome b155t1/2 1—2 msec) in a reaction which is antimycin-A-sensitive. Cytochrome b50 becomes reduced following a flash with a t1/2 1–1.5 msec probably via the primary electron acceptor (Em7.0∼−20 mV) of the reaction-center bacteriochlorophyll. Antimycin A stimulates the extent of cytochrome b50 reduction following a 200-μsec xenon flash but has no detectable effect following a 20-nsec laser flash.3A working model for electron transfer in Rps. spheroides based on the measured thermodynamic and kinetic properties is presented.
Article
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, , 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.
Article
The midpoint potentials of the primary electron acceptors in chromatophores from Rhodopseudomonas spheroides and Chromatium have been studied by titrating the laser-induced P605 and cytochrome c oxidations, respectively. Both midpoint potentials are pH dependent (60 mV/pH unit).o-Phenanthroline shifts the midpoint potentials of the primary acceptors, by +40 mV in Rps spheroides and +135 mV in Chromatium. A similar though less extensive change in midpoint potential was observed in the presence of batho-phenanthroline, but not with 8-hydroxyquinoline. The shifted midpoints retain the same dependence on pH.Some of the effects of o-phenanthroline can be explained by assuming that it chelates the reduced form of the primary electron acceptor. This suggests the presence in the primary electron acceptor of a metal chelated by o- and batho-phenanthroline.In Rps spheroides chromatophores o-phenanthroline inhibits the laser- and flash-induced carotenoid shift at all redox potentials, stimulates the laser-induced P605 oxidation at redox potentials between +350 and +420 mV and slows the decay of the laser-induced cytochrome c oxidation below +180 mV. These effects show that o-phenanthroline may have more than one site of action.
) in Electron Transport and Coupled Energy Transfer in Biological Systems
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