Kinetics of cytochrome b reduction in submitochondrial particles

(1) In agreement with Eisenbach and Gutman (Eisenbach, M. and Gutman, M. (1975) Eur. J. Biochem. 52, 107–116) the reduction of cytochrome b in beef-heart submitochondrial particles by succinate in the presence of antimycin was found to be biphasic, the relative amounts of fast and slow phases being dependent on the redox state of a component located on the oxygen side of the antimycin block. (2) HQNO in a concentration sufficiently large to saturate the specific antimycin- and HQNO-binding sites can substitute for antimycin in these experiments. (3) The rate of the slow phase of the reduction of cytochrome b is decreased under anaerobic conditions and after pretreatment with 2,3-dimercaptopropanol (BAL). (4) In the presence of antimycin and cyanide, cytochrome b-562 is, to some extent, preferentially reduced in the rapid phase and b-566 in the slow phase. (5) The previously proposed regulatory effects of redox-sensitive components X and Y on the redox level and reduction kinetics, respectively, of cytochrome b are ascribed to the role of the Fe-S protein, when it is oxidized, in producing the reductant of cytochrome b by oxidation of QH₂, and by the fact that when QH₂ is bound to it, the reduced Fe-S protein cannot be oxidized by its natural oxidant, cytochrome c₁.     

The effect of pH,ubiquinone depletion and myxothiazol on the reduction kinetics of the prosthetic groups of ubiquinol: Cytochrome c oxidoreductase

(1) The kinetics of the reduction by duroquinol of the prosthetic groups of QH₂: cytochrome c oxidoreductase and of the formation of ubisemiquinone have been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) The formation of the antimycin-sensitive ubisemiquinone anion parallels the reduction of both high-potential and low-potential cytochrome b-562. (3) The rates of reduction of both the [2Fe-2S] clusters and cytochromes (c + c₁) are pH dependent. There is, however, a pH-dependent discrepancy between their rate of reduction, which can be correlated with the difference in pH dependencies of their midpoint potentials. (4) Lowering the pH or the Q content results in a slower reduction of part of the [2Fe-2S] clusters. It is suggested that one cluster is reduced by a quinol/semiquinone couple and the other by a semiquinone/quinone couple. (5) Myxothiazol inhibits the reduction of the [2Fe-2S] clusters, cytochrome c₁ and high-potential cytochrome b-562. (6) The results are consistent with a Q-cycle model describing the pathway of electrons through a dimeric QH₂: cytochrome c oxidoreductase.     

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     

Changes in oxidation-reduction potential of cytochrome b observed in the presence of antimycin A

The cytochrome b of sonic particles of mitochondria or the isolated segment of the respiratory chain containing cytochromes b and c₁ (Complex III) was 80–95% reducible with Q₁H₂ (ubiquinol-5) in the presence of antimycin plus selected electron acceptors added externally (i.e., oxidants which reacted preferentially with respiratory components on the oxygen side of the point of inhibition by antimycin) such as oxygen or ferricyanide depending on whether sonic particles or isolated Complex III was used. In contrast, less than 40% of the cytochrome b was reduced by Q₁H₂ in the absence of either antimycin or the external electron acceptor. In the presence of antimycin ascorbate or mercaptoethanol, which behaved as mild reducing agents, completely inhibited the reduction of cytochrome b by Q₁H₂.     

The photosynthetic electron transfer chain of Chromatium vinosum chromatophores flash-induced cytochrome b reduction

Reduction of a cytochrome b following excitation by a single, short, near-saturating light flash has been demonstrated in Chromatium vinosum chromatophores. The extent of reduction is increased by addition of antimycin. The cytochrome has an α-band maximum at 562 nm in the presence of antimycin.The cytochrome b reduction is most readily observed in the presence of antimycin at high redox potential when cytochrome c-555 is oxidised before excitation. Under these conditions the half-time for reduction is about 20 ms, and the extent is about 0.5 mol of cytochrome b reduced per mol of reaction center oxidised. This extent of reduction is observed on the first flash-excitation from the dark-adapted state, and there was no indication that the reaction center quinone acceptor complex acted as a two-electron accumulating system. With cytochrome c-555 reduced before excitation, the extent of cytochrome b reduction is approximately halved. The factors which result in substoichiometric cytochrome b reduction are not yet understood.Agents which appear to inhibit primary acceptor oxidation by the secondary acceptor (UHDBT, PHDBT, DDAQQ, HOQNO, o-phenanthroline), inhibit reduction of the cytochrome b. DBMIB inhibits cytochrome b reduction but does not appear to inhibit primary acceptor oxidation.These observations confirm that a cytochrome b receives electrons delivered from the primary acceptor complex, and indicate that the photoreduced cytochrome b is reoxidised via an antimycin-sensitive pathway.     

The site of inhibition by 5,5′-dithiobis(2-nitrobenzoate) in ubiquinol:Cytochrome c oxidoreductase

In 5,5′-dithiobis(2-nitrobenzoate) (DTNB)-treated succinate:cytochrome c reductase, the electron transfer from duroquinol to cytochrome c is inhibited due to the fact that the Rieske Fe-S cluster and, consequently, cytochrome c, are no longer reducible by substrate. The finding that, after this treatment, cytochrome b is still reducible by substrate in the absence of antimycin, but not in its presence, is consistent with a Q-cycle mechanism for the electron transfer through QH₂:cytochrome c oxidoreductase. The inhibitory effect of DTNB and its effect on the EPR spectrum of the [2Fe-2S] cluster suggest that it prevents either the binding of ubiquinone in the vicinity of this cluster or the interaction between the Fe-S protein and a ubiquinone-binding protein.     

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.     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

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.     

Cytochrome b reduction by hexaammineruthenium in mitochondria and submitochondrial particles: Evidence for heme b-562 localization at the M-side of the mitochondrial membrane

In the presence of ascorbate, hexaamineruthenium mediates rapid reduction of cytochrome b-562 in submitochondrial particles but not in mitochondria. The reaction is obsreved in the combined presence of antimycin (or funiculosin) and myxothiazol, which implies direct interaction of Ru(NH₃)²⁺₆ with b cytochrome(s). We assume that contrary to previous conclusions (Case and Leigh (1976) Biochem. J., 160, 769-783) redox centre of at least one of the oxidized cytochromes b, most probably of b-562, is exposed to the M-aqueous phase.     

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 triphasic reduction of cytochrome b in the succinate-cytochrome c reductase

In the succinate-cytochrome c reductase, the reduction of cytochrome b has been found to be triphasic: an initial rapid partial reduction was followed first by a rapid oxidation and then finally by a slow reduction. The initial reduction of cytochrome b was faster than that of cytochrome c₁ and the final slow reduction of cytochrome b began when cytochrome c₁ reduction was approaching completion. In presence of the inhibitors antimycin A or HQNO the reduction of cytochrome b became monophasic. Hysteresis or a kinetic cooperative effect of a factor controlling cytochrome b oxidation has been suggested as a possible explanation for the triphasic reduction of cytochrome b.     

Funiculosin: An antibiotic with antimycin-like inhibitory properties

The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg²⁺-ATP particles (0.4–0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc₁. Funiculosin also induces the oxidation of cytochromes cc₁ and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

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.     

The pathway of cyclic electron transport in chromatophores of Chromatium vinosum. Evidence for a Q-cycle mechanism

Chromatophores of the purple sulfur bacterium Chromatium vinosum were shown to contain a cytochrome similar to cytochrome c₁ and two b cytochromes. Cytochrome b can be accumulated in the reduced form upon illumination at an ambient redox potential of +415 mV in the presence of the electron transport inhibitors antimycin A or HOQNO. The reductions of cytochrome b, of the high-potential cytochrome c₅₅₅ and of the primary electron donor P-870 are all inhibited by myxothiazol. Dark-adapted C. vinosum chromatophores show little cytochrome b reduction on the first flash. Considerable cytochrome b reduction (1 cytochrome b:8 P-870 present) is observed on the second flash. This observation and the 1:1 stoichiometry observed between cytochrome b reduction and P-870⁺ reduction after the second flash support a Q-cycle model for cyclic electron flow in C. vinosum.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.     

The pathway of electrons through OH2:cytochrome c oxidoreductase studied by pre-steady -state kinetics

The kinetic behaviour of the prosthetic groups and the semiquinones in in QH2:cytochrome c oxidoreductase has been studied using a combination of the freeze-quench technique, low-temperature diffuse-reflectance spectroscopy, EPR and stopped flow. (2) In the absence of antimycin, cytochrome b-562 is reduced in two phases separated by a lag time. The initial very rapid reduction phase, that coincides with the formation of the antimycin-sensitive Qin, is ascribed to high-potential cytochrome b-562 and the slow phase to low-potential cytochrome b-562. the two cytochromes are present in a 1:1 molar ratio. The lag time between the two reduction phases decreases with increasing pH. Both the [2 Fe-2S] clusters and cytochrome c1 are reduced monophasically under these conditions, but at a rate lower than that of the initial rapid reduction of cytochrome b-562. (3) In the presence of antimycin and absence of oxidant, cytochrome b-562 is still reduced biphasically, but there is no lag between the two phases. No Qin is formed and both the Fe-S clusters and cytochrome c1 are reduced biphasically, one-half being reduced at the same rate as in the absence of antimycin and the other half 10-times slower. (4) In the presence of antimycin and oxidant, the recently described antimycin-insensitive species of semiquinone anion, Qout (De Vries, S., Albracht, S.P.J., Berden, J.A. and Slater, E.C. (1982) J. Biol. Chem. 256, 11996-11998) is formed at the same rate as that of the reduction of all species of cytochrome b. In this case cytochrome b is reduced in a single phase. (5) The reversible change of the line shape of the EPR spectrum of the [2Fe-2S] cluster 1 is caused by ubiquinone bound in the vicinity of this cluster. (6) The experimental results are consistent with the basic principles of the Q cycle. Because of the multiplicity, stoicheiometry and heterogeneous kinetics of the prosthetic groups, a Q cycle model describing the pathway of electrons through a dimeric QH2:cytochrome c oxidoreductase is proposed.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.