The role of cytochrome b-566 in the electron-transfer chain of Rhodopseudomonas sphaeroides

Spectrophotometric, kinetic, thermodynamic and stoichiometric properties of the low-potential b-type cytochrome of chromatophores from Rhodopseudomonas sphaeroides are reported. Cytochrome b-566 has a double α-band with maxima at 559 and 566 nm. Resolution of the spectrum by full-spectral redox potentiometry showed no indication that the two peaks represent more than one component. The component titrated with E_m,7 ≈ ?80 ± 10 mV. By appropriate choice of wavelength pairs and by subtraction of the contribution due to other components, the kinetics of cytochrome b-566 absorbance changes following flash excitation have been resolved from those of other components. Time-resolved flash spectra corrected for the contributions of other components are consistent with the behavior of both peaks of the α-band as a single kinetic species. The kinetics of cytochrome b-566 in the presence of antimycin show that the reduction of this cytochrome occurred only if cytochrome b-561 was reduced before the flash, either chemically, by poising the ambient redox potential (E_h) below the E_m of cytochrome b-561 (E_m,7 ≈ 50 mV), or photochemically at higher redox potentials by a previous flash. The rate of reduction of cytochrome b-566 varied with E_h. At low E_h (approx. 0 mV) reduction on the first flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1.25}\text{ms}$; at high E_h (approx. 180 mV) reduction on the second flash showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 10}\text{ms}$. In the absence of antimycin at E_h ≈ 0 mV, cytochrome b-566 was observed to become rapidly reduced ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 500 μ}\text{s}$) and then reoxidized ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 2}\text{ms}$) after a single flash. At higher redox potentials (E_h > 80 mV) no kinetic changes which could be unambiguously attributed to cytochrome b-566 were observed following a single flash. The results are interpreted in terms of a Q-cycle mechanism in which the reductant for cytochrome b-566 is the semiquinone formed on oxidation of ubiquinol from the quinone pool. The oxidation of the ubiquinol occurs by a concerted reaction in which one electron is accepted by the Rieske-type FeS center and the other by cytochrome b-566. We suggest that the kinetic characteristics may indicate a pathway for reduction of the b-type cytochromes in which cytochrome b-566 is the immediate electron acceptor and donates to cytochrome b-561 in a serial pathway. The experimental results in the presence of antimycin are compared with data from a computer simulation of the thermodynamic behavior of the chain, and the computer model is shown to provide an excellent fit.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(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^?_I) and the secondary acceptor ubiquinone (Q_II) 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 E_h(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 E_m(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^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, 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^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(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 E_h 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     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x 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 (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max 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.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 ± 20 mM^?1 · cm^?1. The molecular weight based on protein and chromophore assays was found to be 1.5 · 10⁵; the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Flash-induced turnover of the cytochrome bc1 complex in chromatophores of Rhodobacter capsulatus: binding of Zn decelerates likewise the oxidation of cytochrome b, the reduction of cytochrome c1 and the voltage generation

The effect of Zn²⁺ on the rates of electron transfer and of voltage generation in the cytochrome bc₁ complex (bc₁) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn²⁺ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme b_h (in the absence of Zn²⁺ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c₁ reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn²⁺ <100 μM the reduction of heme b_h remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc₁ turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme b_h is the main electrogenic reaction in the cytochrome bc₁ complex, (2) imply an allosteric link between the site of heme b_h oxidation and the site of cytochrome c₁ reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc₁ complex.     

Molar extinction coefficients and other properties of an improved reaction center preparation from Rhodopseudomonas viridis

Reaction centers have been purified from chromatophores of Rhodopseudomonas viridis by treatment with lauryl dimethyl amine oxide followed by hydroxyapatite chromatography and precipitation with ammonium sulfate. The absorption spectrum at low temperature shows bands at 531 and 543 nm, assigned to two molecules of bacteriopheophytin b. The 600 nm band of bacteriochlorophyll b is resolved at low temperature into components at 601 and 606.5 nm. At room temperature the light-induced difference spectrum shows a negative band centered at 615 nm, where the absorption spectrum shows only a weak shoulder adjacent to the 600 nm band. The fluorescence spectrum shows a band at 1000 nm and no fluorescence corresponding to the 830 nm absorption band. Two molecules of cytochrome 558 and three of cytochrome 552 accompany each reaction center. The differential extinction coefficient (reduced minus oxidized) of cytochrome 558 at 558 nm was estimated as 20 ± 2 mM^?1 · cm^?1 through a coupled reaction with equine cytochrome c. The extinction coefficient of reaction centers at 960 nm was determined to be 123 ± 25 mM^?1 · cm^?1 by measuring the light-induced bleaching of P-960 and the coupled oxidation of cytochrome 558. The corresponding extinction coefficient at 830 nm is 300 ± 65 mM^?1 · cm^?1. The absorbance ratio $\text{a}{}_{\mathrm{<mtext>280</mtext><mtext>nm</mtext>}}\text{a}{}_{\mathrm{<mtext>830</mtext><mtext>nm</mtext>}}$ in our preparations was 2.1, and there was 190 kg protein per mol of reaction centers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major components of apparent molecular weights 31 000, 37 000 and 41 000.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas Sphaeroides

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 anti-mycin, 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 reduction 3- to 4-fold under certain if not all conditions.4. A maximum of approximately 0.6 cytochrome b-560 (E_m(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 (Q_z) 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 Q_z, 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 (Q_II) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Q_z, respectively.     

A thermodynamic characterisation of the cytochromes of chromatophores from Rhodopseudomonas capsulata

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

Differential extraction and structural specificity of specialized ubiquinone molecules in secondary electron transfer in chromatophores from Rhodopseudomonas sphaeroides, Ga

In chromatophores from the facultative photosynthetic bacterium, Rhodopseudomonas sphaeroides, Ga, the function of ubiquinone-10 (UQ-10) at two specialized binding sites (Q_B and Q_Z) has been determined by kinetic criteria. These were the rate of rereduction of flash-oxidized [BChl]₂⁺ through the back reaction, or the binary pattern of cytochrome b₅₆₁ (for the Q_b site), and the rapid rate of rereduction of flash-oxidized cytochrome c, or the relative amplitude of the antimycin-sensitive Phase III ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{~ 1.5}\text{ms}$) of the carotenoid spectral shift induced by a single turnover flash at E_h ~ 100 mV (for the Q_Z site). The phenomenon associated with the two binding sites behaved differently on extraction of UQ from lyophilized chromatophores using isooctane. By this selective extraction procedure it has been possible to show that UQ-10 molecules are required at different concentrations in the membrane for specific redox events in secondary electron transfer. The reduction of cytochrome b occurs in particles which no longer show the phenomena associated with Q_Z, but still possess a large proportion of Q_b, while rapid rereduction of flash-oxidized cytochrome c requires an additional complement of UQ-10 (Q_Z). Extracted particles lacking Q_Z and a large amount of Q_B have been reconstituted with different UQ homologs (UQ-1, UQ-3, and UQ-10). Specific redox events have been studied in reconstituted particles. All UQ homologs act as secondary acceptors from the reaction center; UQ-3 and UQ-10, but not UQ-1, are also able to reconstitute the function of Q_Z as electron donor to cytochrome c. Only UQ-10, however, is able to restore normal rates of the overall cyclic electron transfer induced by a train of flashes, and maximal rates of the light-induced ATP synthesis. The results are interpreted in terms of Q-cycle mechanisms in which quinone and quinol at both the Q_Z and Q_b sites are in rapid equilibrium with the quinone pool.     

Isolation and Purification of Cytochrome b561 from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ was removed from chromatophores of a photoanaerobicallygrown Rhodopseudomonas sphaeroides by deoxycholate-cholate andTriton X-100 treatments of the chromatophores. The cytochromewas purified by ammonium sulfate fractionation and gel filtration.Its molecular weight was 45,000 (45 kD) and it was composedof three subunits with molecular weights of 23 kD, 19 kD andless than 6 kD. The cytochrome preparation had absorption maximaat 414 nm in the oxidized form, and at 428, 530 and 561 nm inthe reduced form. Its pi was 4.8. The midpoint potential ofthis cytochrome was 153 mV at pH 7.0. The compound was autooxidizable,and it had cytochrome c oxidase activity. (Received May 16, 1983; Accepted September 8, 1983)     

The interaction of the reaction center secondary quinone with the ubiquinone-cytochrome c2 oxidoreductase in Rhodopseudomonas sphaeroides chromatophores

(1) Two populations of reaction centers in the chromatophore membrane can be distinguished under some conditions of initial redox poise (300 mV < E_h < 400 mV): those which transfer a reducing equivalent after the first flash from the secondary quinone (Q_II) of the reaction center to cytochrome b of the ubiquinone-cytochrome c₂ oxidoreductase; and those which retain the reducing equivalent on Q^?_II until a second flash is given. These two populations do not exchange on a time scale of tens of seconds. (2) At redox potentials higher than 400 mV, Q^?_II generated after the first flash is no longer able to reduce cytochrome b-560 even in those reaction centers associated with an oxidoreductase. Under these conditions, doubly reduced Q_II generated by a second flash is required for cytochrome b reduction, so that the Q_II effectively functions as a two-electron gate into the oxidoreductase at these high potentials. (3) At redox potentials below 300 mV, although the two populations of Q_II are no longer distinguishable, cytochrome b reduction is still dependent on only part of the reaction center population. (4) Proton binding does not oscillate under any condition tested.     

The role of the quinone pool in the cyclic electron-transfer chain of Rhodopseudomonas sphaeroides A modified Q-cycle mechanism

(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 c₂ oxidoreductase catalyses a concerted reaction in which one electron is transferred to a high-potential chain containing cytochromes c₁ and c₂, 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 c₁ and c₂, 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 ix 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 c₂ oxidoreductase complex, and the net oxidation of one ubiquinol/complex. (7) The antimycin-sensitive reduction of cytochrome c₁ and c₂ 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.     

The kinetics of flash-induced electron flow in bacteriochlorophyll-less membranes of Rhodopseudomonas sphaeroides reconstituted with reaction centres

Membranes isolated from aerobically grown mutants 01 and PM8bg II-15 of Rhodopseudomonas sphaeroides lack reaction centres. Incorporation of purified reaction centres into these membranes can be achieved by mixing the protein and membranes in 1% sodium cholate with added soybean phospholipid and removing the cholate by dialysis.The kinetics of light-stimulated electron flow in these reconstituted membranes have been examined and compared with those observed in chromatophore membranes isolated from photosynthetically grown R. sphaeroides. Following a single saturating flash, reconstituted reaction centres become photo-oxidised, and about 60% are re-reduced within about 200 ms by cytochrome c2 in the 01 membrane. Cytochrome c2 photo-oxidation is biphasic, the half-time of the first fase being faster than 20 μs. The second phase is variable and can be as slow as 60 ms. A cytochrome b in the membrane becomes photoreduced with a half-time of 27 ms. Electron flow between cytochromes b and c2 is slow and appears only partially sensitive to antimycin A.Using membranes from the reaction centre-less mutant PM8bg II-15 similar reconstitution measurements were performed. The resulting kinetic measurements showed that fast cytochrome b photoreduction and cytochrome c2 photo-oxidation occurred.The absorbance change at 560 minus 570 nm induced by steady-state illumination of 01 membranes reconstituted with reaction centres was measured at a range of ambient potentials; the reaction was abolished at oxidation-reduction potentials below 0 mV. The change was approximately halved at +50 mV, indicating that cytochrome b₊₅₀ is the recipient of electrons from the reconstituted reaction centres.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

In situ characterisation of photosynthetic electron transport in Rhodopseudomonas capsulata

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.     

A method for in situ characterization of b- and c-type cytochromes in Escherichia coli and in Complex III from beef heart mitochondria by combined spectrum deconvolution and potentiometric analysis

An analytical technique for the in situ characterization of b- and c-type cytochromes has been developed. From evaluation of the results of potentiometric measurements and spectrum deconvolutions, it was concluded that an integrated best-fit analysis of potentiometric and spectral data gave the most reliable results. In the total cytochrome b content of cytoplasmic membranes from aerobically grown Escherichia coli, four major components are distinguished with α-band maxima at 77 K of 555.7, 556.7, 558.6 and 563.5 nm, and midpoint potentials at pH 7.0 of 46, 174, ?75 and 187 mV, respectively. In addition, two very small contributions to the α-band spectrum at 547.0 and 560.2 nm, with midpoint potentials of 71 and 169 mV, respectively, have been distinguished. On the basis of their spectral properties they should be designated as a cytochrome c and a cytochrome b, respectively. In Complex III, isolated from beef heart mitochondria, five cytochromes are distinguished: cytochrome c₁ (Λ_m(25°C) = 553.5 nm; E′₀ = 238 mV) and four cytochromes bΛ_m(25°C) = 558.6, 561.2, 562.1, 566.1 nm and E′₀ = ?83, 26, 85, ?60 mV).     

Purification and partial characterization of cytochrome b5 from Tetrahymena pyriformis

With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b₅. However, it was reducible by NADH in the presence of NADH-cytochrome b₅ reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b₅ of a ciliated protozoan, Tetrahymena pyriformis.     

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

Mechanism of nitrite reduction to nitrous oxide in a photodenitrifier,Rhodopseudomonas sphaeroide forma sp. denitrificans

The mechanism of anaerobic reduction of NO₂^? to N₂O in a photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans, was investigated. With ascorbate-reduced phenazine methosulfate (PMS) as the electron donor, the nitrite reductase of this photodenitrifier reduced NO₂^? to NO and a trace amount of N₂O. With dithionite-reduced benzyl viologen as the electron donor, the major product of NO₂^? reduction was NH₂OH, and a trace amount of N₂O was also produced. The nitrate reductase itself had no NO reductase activity with ascorbate-reduced PMS. It was concluded that the essential product of NO₂^? reduction by the purified nitrite reductase is NO. Chromatophore membranes stoichiometrically produced N₂O from NO₂^? with any electron donor, such as dithionite-redduced benzyl viologen, ascorbate-reduced PMS or NADH/FMN. The membranes also contrained activity of NO reduction of N₂O with either ascorbate-reduced PMS or duroquinol. The NO reductase activity with duroquinol was inhibited by antimycin A. Stoichiometric production of N₂O from N₂^? also was observed in the reconstituted NO₂^? reduction system which contained the cytochrome bc₁ complex, cytochrome c₂, the nitrite reductase and duroquinol as the electron donor. The preparation of the cytochrome bc₁ complex itself contianed NO reductase activity. From these results the mechanism of NO₂^? reduction to N₂O in this photodenitrifier was determined as the nitrite reductase reducing NO₂^? to NO with electrons from the cytochrome bc₁ complex, and NO subsequently being reduced, without release, to N₂O with electrons from the cytochrome bc₁ complex by the NO reductase, which is closely associated with the complex.