A new electrogenic step in the ubiquinol:cytochrome c2 oxidoreductase complex of Rhodopseudomonas sphaeroides

Myxothiazol, an inhibitor of the ubiquinol oxidase site of the ubiquinol:cytochrome c2 oxidoreductase complex, has been shown in the present work to inhibit a part of the electrogenic process indicated by phase III of the carotenoid change, in addition to the part of the change inhibited by antimycin. This finding shows that there is an antimycin-insensitive, but myxothiazol-sensitive portion of the slow phase, which indicates the existence of an electrogenic event within the ubiquinol:cytochrome c2 oxidoreductase complex, in addition to that linked to oxidation of cytochrome b-561 which has been previously characterized. Redox titrations show that the appearance of the new electrogenic step is correlated with the amount of cytochrome b-561 available in the oxidized form before the flash. The rate of the antimycin-insensitive and myxothiazol-sensitive portion of the carotenoid change correlates well with the rate of reduction of cytochrome b-561. No carotenoid change associated with reduction of cytochrome b-566 was seen. These findings suggest that the newly identified electrogenic process is linked to electron transfer between cytochrome b-566 and b-561. Calculations of the contribution of this new electrogenic step to the total electrogenic event within the complex show that electrons passing from cytochrome b-566 to cytochrome b-561 pass about 35-50% of the distance across the whole membrane.     

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.     

Activity of reduced ubiquinone: Cytochrome c oxidoreductase with various ubiquinol-isoprenologues as substrate and corresponding inhibitory effect of antimycin in yeast

1. Reduced ubiquinones-1, -2, -3, -4 and -6 were used as substrates for ubiquinol: cytochrome c oxidoreductase.2. The portion of antimycin-sensitive activity depends on the concentration of ubiquinol and on the pH. Only reduced ubiquinone-2 and reduced ubiquinone-3 show high activities the main part of which is sensitive to antimycin.3. The antimycin effect curve of ubiquinol: cytochrome c oxidoreductase is linear in shape with reduced ubiquinone-2 as substrate but sigmoidal with reduced ubiquinone-3 and succinate. Ubiquinol-3: cytochrome c oxidoreductase activity contains a portion scarcely affected by antimycin. About 300 pmoles of antimycin per mg protein, enough to inhibit succinate, NADH- and reduced ubiquinone-2:cytochrome c oxidoreductase almost totally, affect ubiquinol-3: cytochrome c oxidoreductase to only about 80% and another 300 pmoles of antimycin are needed for the next 10% of inhibition.4. The activities of succinate- and NADH: cytochrome c oxidoreductase are stimulated by ubiquinones-2 and -3. The shapes of the inhibition curves by antimycin of the stimulated activities are sigmoidal. About twice the amount of antimycin is necessary to inhibit stimulated activities to the same value as the unstimulated.5. The non-ionic detergent Lubrol WX is not effective in stimulating enzymatic activities. However, in the presence of 0.6 M sorbitol, it converts the linear antimycin effect curve with reduced ubiquinone-2 as substrate, into sigmoidal.6. NADH- and succinate: cytochrome c oxidoreductase activities and reduced ubiquinone-2 and reduced ubiquinone-3: cytochrome c oxidoreductase activities become deactivated with increasing concentrations of the non-ionic detergent Lubrol WX. The activity with reduced ubiquinone-2 as substrate is less resistant to the action of the detergent than with reduced ubiquinone-3. The b-cytochromes do not become CO-reactive by this treatment.7. Deoxycholate in low concentrations does not stimulate ubiquinol: cytochrome c oxidoreductase activity. It converts the inhibition curve by antimycin from sigmoidal to linear with increasing concentrations of the detergent with all substrates tested. The amount of antimycin needed for 90% inhibition of reduced ubiquinone activities is about the same under these conditions as with succinate, NADH or reduced ubiquinol in untreated particles.8. The results are discussed with respect to the theories of the electron transport mechanism and of the inhibition by antimycin of the electron flow through the bc₁-segment of the respiratory chain in beef heart.     

A myxothiazol-sensitive Q-binding protein isolated from Chromatium vinosum

A quinol: ferricytochrome c oxidoreductase has been isolated from chromatophores of Chromatium vinosum by two procedures, involving extraction by bile salts and methanol, respectively. The steady-state kinetics indicate a random mechanism, with a K_m for 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol of 1.1 μM and for the acceptor cytochrome c 1.75 μM. The enzyme is inhibited by myxothiazol, competitively with respect to quinol, with a K_i of about 2.3 μM. The protein reacts with ubiquinol produced by the succinate: Q oxidoreductase in submitochondrial particles or isolated succinate: cytochrome c reductase and can partially restore activity to myxothiazol-inhibited, antimycin-sensitive ubiquinol: cytochrome c oxidoreductase. The protein is considered to be analogous to the postulated myxothiazol-sensitive Q-binding protein in ubiquinol: cytochrome c oxidoreductase.     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

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.     

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     

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 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.     

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.     

Electron transport in the membrane of lutoids from the latex ofHevea brasiliensis

1. An antimycin-insensitive NADH-cytochromec oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex ofHevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH.2. Twob-type cytochromes are present in the membranes. Cytochromeb₅₆₃ is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 °K. A second cytochrome, cytochromeb₅₆₁, seems to be reducible by hydrosulfite only.3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochromeP-450 could not be demonstrated.4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Parallel electron donation pathways to cytochrome cz in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c_z bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c_z. It was also shown that the rereduction rate of cytochrome c_z by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Kinetics of flash-induced electron transfer between bacterial reaction centres,mitochondrial ubiquinol: Cytochrome c oxidoreductase and cytochrome c

Ascorbate-reduced horse heart cytochrome c reduces photo-oxidized bacterial reaction centres with a second-order rate constant of (5–8) · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. In the absence of cytochrome c, the cytochrome c₁ in the ubiquinol:cytochrome c oxidoreductase is oxidized relatively slowly (k = 3.3 · 10⁵ M^?1 · s^?1). Ferrocytochrome c binds specifically to ascorbate-reduced reductase, with a K_d of 0.6 μM, and only the free cytochrome c molecules are involved in the rapid reduction of photo-oxidized reaction centres. The electron transfer between ferricytochrome c and ferrocytochrome c₁ of the reductase is rapid, with a second-order rate constant of 2.1 · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. The rate of electron transfer from the Rieske iron-sulphur cluster to cytochrome c₁ is even more rapid. The cytochrome b of the ubiquinol:cytochrome c oxidoreductase can be reduced by electrons from the reaction centres through two pathways: one is sensitive to antimycin and the other to myxothiazol. The amount of cytochrome b reduced in the absence of antimycin is dependent on the redox potential of the system, but in no case tested did it exceed 25% of the amount of photo-oxidized reaction centres.     

Slow electrogenic events in the cytochrome bc1-complex of Rhodobacter sphaeroides. The electron transfer between cytochrome b hemes can be non-electrogenic.

The flash-induced formation of transmembrane electric potential differences (measured by carotenoid bandshift) and redox changes of cytochrome bh (b561) were monitored spectrophotometrically in Rb. sphaeroides chromatophores in a pH range from 7.5 to 10.0. It is shown that in the presence of antimycin A and at pH less than 8.3 the myxothiazol-sensitive, antimycin-insensitive component of the carotenoid bandshift is kinetically coupled to cytochrome bh reduction. The kinetics of both processes can be described by a single exponent with a rise time of about 10 ms. Alkalization of the medium (8.3 less than or equal to pH less than or equal to 9.2) causes the appearance of an additional constituent in this phase of the carotenoid response with the rise time varying in the range of 100-300 ms. With a further pH increase (pH greater than 9.2), the electrogenic constituent, kinetically linked to cytochrome bh reduction, diminishes. The obtained data are discussed within the framework of the scheme, assuming that the electron transfer between bl and bh hemes in the bc1 complex is, under certain conditions, accompanied by proton transfer in the same direction.     

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).     

Mitochondrial cytochrome b-c complex: its oxidation-reduction components and their stoichiometry.

A cytochrome b-c₁ complex was isolated from pigeon breast muscle mitochondria and purified to a content of 3 nmol of cytochrome c₁ per milligram of protein. Anaerobic suspensions of the preparation were titrated with reducing equivalents (NADH) and oxidizing equivalents (ferricyanide). The oxidation-reduction components of the complex were measured by the number of reducing equivalents accepted or donated per cytochrome c₁ and compared with the stoichiometries of the known redox components as measured by independent methods. The preparation accepts or donates 5.2 ± 0.3 equivalents per cytochrome c₁, while the measured content of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₅, Rieske iron-sulfur protein, ubiquinone, and succinate dehydrogenase accounts for 5.0 ± 0.2 equivalents per cytochrome c₁. It is concluded that there are no unknown redox components in the cytochrome b-c₁ complex. The cytochrome b-c₁ complex (energy transduction site 2) appears to be a structural unit containing equal amounts of cytochrome c₁, cytochrome b₅₆₁, cytochrome b₅₆₆, and the Rieske iron-sulfur protein.     

Characterization of cytochrome b in the isolated ubiquinol-cytochrome c2 oxidoreductase from Rhodopseudomonas sphaeroides GA

Extinction coefficients for cytochrome b and c₁ in the isolated cytochrome bc₁ complex from Rhodopseudomonas sphaeroides GA have been determined. They are 25 mM^?1.cm^?1 at 561 nm for cytochrome b and 17.4 mM^?1.cm^?1 at 553 nM for cytochrome c₁ for the difference between the reduced and the oxidized state. Cytochrome b is present in two forms in the complex. One form has an E_m7 of 50 mV, an α-peak of 557 nm at liquid N₂ temperature and of 561 nm at RT, which is red-shifted by antimycin A. The other form has an E_m7 of ?90 mV, a double α-peak of 555 and 561 nm at liquid N₂ temperature corresponding to 559 and 566 nm at RT. The absorption at 566 nm is red-shifted by myxothiazol. The two shifts are independent of each other. Both midpoint potentials of cytochromes b are pH-dependent. The redox center compositions of the cytochrome bc₁ complexes from Rhodopseudomonas sphaeroides and from mitochondria are identical.     

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.     

Membranes of Rhodopseudomonas sphaeroides VII. Photochemical properties of a fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes

Previous pulse-chase studies have shown that bacteriochlorophyll a-protein complexes destined eventually for the photosynthetic (chromatophore) membrane of Rhodopseudomonas sphaeroides appear first in a distinct pigmented fraction. This rapidly labeled material forms an upper band when extracts of phototrophically grown cells are subjected directly to rate-zone sedimentation. In the present investigation, flash-induced absorbance changes at 605 nm have demonstrated that the upper fraction is enriched two-fold in photochemical reaction center activity when compared to chromatophores; a similar enrichment in the reaction center-associated B-875 antenna bacteriochlorophyll complex was also observed. Although b- and c-type cytochromes were present in the upper pigmented band, no photoreduction of the b-type components could be demonstrated. The endogenous c-type cytochrome (E_m = +345 mV) was photooxidized slowly upon flash illumination. The extent of the reaction was increased markedly with excess exogenous ferrocytochrome c but only slightly in chromatophores. Only a small light-induced carotenoid band shift was observed. These results indicate that the rapidly labeled fraction contains photochemically competent reaction centers associated loosely with c-type and unconnected to b-type cytochrome. It is suggested that this fraction arises from new sites of cytoplasmic membrane invagination which fragment to form leaky vesicles upon cell disruption.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.