Structure and function of the cytochrome bc1 complex of mitochondria and photosynthetic bacteria.

Progress has recently been made in the understanding of the function of the cytochrome bc1 complex and related proteins in the context of recent structural information. The structures support many features that were predicted from sequence analysis and biophysical studies, but contain some surprises. Most dramatically, it is apparent that the iron-sulfur protein can take up different positions in different crystals, suggesting a novel mechanism for electron transfer through domain movement. Evidence from studies of mutant strains, in which the function of the sites or the binding of inhibitors is perturbed, has provided clues about the mechanism.

Binary oscillations in the rate of reoxidation of the primary acceptor of photosystem II.

The decay of fluorescence yield following each of a series of saturating laser flashes has been used to monitor the kinetics of reoxidation of the primary acceptor of Photosystem II under conditions of varied redox potential. 1. In dark-adapted chloroplasts, a damped binary oscillation as a function of flash number was observed in the kinetics of the decay of the fluorescence yield. The decay was faster on odd than on even-numbered flashes. 2. In the presence of low concentrations of 1,4-benzoquinone, the oscillation was more marked, and over the range approx 200--350 mV, independent of redox potential. The decay following flash 1 under these conditions had a half-time of approx. 200--400 microseconds. The decay following flash 2 was decelerated; the initial rate was up to 10-fold slower than after flash 1. 3. We suggest that the kinetics following a single flash reflect the rate of the reaction Q-B leads to QB-, and following the second flash, Q-B- leads to QB2-. Benzoquinone at low concentrations oxidises a residual fraction of B- which is usually reduced in the dark before the flash sequence. 4. A faster component in the decay (t u/2 approximately 140 microseconds) following the first flash titrated in over the range Eh greater than 350 mV. The binary oscillation was still apparent but delayed by one flash. 5. We discuss the relative redox potentials of the couples B/B- and B-/B2-, and the role of the component which titrates in at Eh greater than 350 mV.

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 alpha-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 initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light.

1. Photophosphorylation was studied in spinach chloroplasts on illumination, from the dark state, with saturating short ("single turnover") flashes of light. 2. At rapid flash rates (100 Hz), phosphorylation began within the first five flashes. The ATPase inhibitor protein appeared to be displaced from its inhibitory site on the ATPase also within five flashes, as deduced from the flash-induced ATPase activity. 3. At slower flash rates, or if the rate of electron transfer were reduced with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), phosphorylation began only after a larger number (50--60) of flashes. The displacement of the ATPase inhibitior protein was similarly delayed. 4. Partial displacement of the inhibitor protein from its inhibitory site on the ATPase (by pretreatment with dithioerythritol) allowed phosphorylation to proceed without a perceptible lag, even in the presence of DCMU. It was concluded that the ATPase inhibitor protein must be displaced on the ATPase before phosphorylation can begin, and that this process is energy dependent. 5. During the flash regime used, release of inhibitor from its inhibitory site seemed to be governed largely by the membrane potential. The light-induced pH gradient seemed to have little effect under these conditions. Our results are not compatible with a direct conformational interaction between the electron transfer chain and the ATPase causing displacement of the inhibitor. 6. The maximal rate of photophosphorylation induced by less than 200 flashes was 0.12--0.15 mol ATP made/mol ATPase per flash. This rate seemed to be limited not be the supply of energy to the ATPase molecules, nor by the maximal turnover capacity of the ATP synthesising system, but by the number of ATPase molecules which were active in synthesis, i.e., which lacked the inhibitor protein. 7. The bound nucleotides of the coupling ATPase exchanged with added nucleotides during single turnover flashes. At high flash rates, exchange began within 5 flashes. The average amount of nucleotide exchanged per flash over 100 flashes was about one tenth the amount of ATP synthesised in each flash. 8. It is concluded that, during phosphorylation, a steady state level of active coupling ATPases is set up. The energy-dependent displacement of the inhibitor protein, and its (energy-independent) relaxation back on to the inhibitory site are the two opposing factors involved in this steady state.

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides.

The pigment content of a B800-850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed. The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800-850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band. The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.

Kinetics of the c-cytochromes in chromatophores from Rhodopseudomonas sphaeroides as a function of the concentration of cytochrome c2. Influence of this concentration on the oscillation of the secondary acceptor of the reaction centers QB.

The oxidation kinetics of Cyt c1 and c2 have been measured in normal chromatophores and in chromatophores fused with liposomes in order to increase the internal volume. The kinetics of Cyt c1 oxidation were found to be dependent on Cyt c2 concentration. The initial rate of Cyt c1 oxidation decreased after fusion by a factor of about two, indicating a process dependent on diffusion. The results do not allow a clear distinction between a diffusion of Cyt c2 along the inner membrane surface or through the inner volume of the vesicle; two- and three-dimensional models are discussed. In contrast to Cyt c1, the kinetics of oxidation of Cyt c2 were not influenced by changes in concentration. It is concluded that reduced Cyt c2 is preferentially bound to the reaction centers. A binary pattern as a function of flash number from the dark-adapted state was measured in the turn-over of the two-electron gate of the reaction center. In chromatophores with more than 0.5 cytochrome c2 molecules per reaction center, this binary pattern titrated out with a midpoint around 340 mV on reduction of the suspension. In experiments with chromatophores with a low Cyt c2 content, or with spheroplast-derived vesicles which had lost Cyt c2, the binary oscillation in the two-electron gate could be observed at much lower potentials. The results suggest that the binding of reduced cytochrome c2 modifies the behavior of the two-electron gate. A model in which reaction center dimers are stabilized by Cyt c2 is proposed to explain the effect.

Electron transport in chromatophores from Rhodopseudomonas sphaeroides GA fused with liposomes.

Chromatophores from Rhodopseudomonas sphaeroides GA were fused with liposomes in order to dilute the components of the cyclic photosynthetic electron-transport chain within the membrane. This dilution led to a decrease in the rate of cytochrome b-561 reduction. The original rates could be restored at potentials around 100 mV (where a large part of the quinone pool is chemically reduced), if ubiquinone was incorporated into the liposomes prior to fusion. Similar dilution effects could be observed in synchronized cultures. The membrane obtained after division contained about twice the amount of phospholipids per reaction center when compared to chromatophores prepared from cells harvested just before division. Chromatophores from synchronized cultures are more uniform with respect to the concentration of the different electron-transport components in the membrane than the membranes from normally grown cells. The kinetic behaviour both of fused chromatophores and of membranes from synchronized cultures are in agreement with a modified Q-cycle model for photosynthetic electron transport in Rps. sphaeroides. The results presented in this paper cannot be explained by postulating the presence of a firmly bound quinone, Qz, in the ubiquinol: cytochrome c2 oxidoreductase, as previously proposed.

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 relationship between delayed fluorescence and the carotenoid shift in chromatophores from Rhodopseudomonas capsulata.

(1) The kinetics of the change in intensity of 1-ms delayed fluorescence following the onset of illumination have been studied in chromatophores of Rhodopseudomonas capsulata and compared with the kinetics of the light-induced carotenoid shift, and of the change in fluorescence yield. The change in intensity of delayed fluorescence, when plotted on a logarithmic scale, followed closely the kinetics of carotenoid change, but not the change in fluorescence yield. (2) The antibiotics valinomycin, nigericin and antimycin, showed qualitatively similar effects on delayed fluorescence and the carotenoid shift, but the effects on fluorescence yield were quite dissimilar. (3) The extent of the carotenoid shift, induced by KCl pulses in the presence of valinomycin was used to calibrate the change as an indicator of membrane potential. The action of nigericin on both carotenoid shift and delayed fluorescence was used to relate the two, and a linear relationship was demonstrated between the logarithm of the intensity of the delayed fluorescence and the extent of the carotenoid shift. This relationship indicated that the intensity of delayed fluorescence was proportional to the exponential of the membrane potential. (4) It is suggested that the electrical component of the transmembrane electrochemical H+ gradient directly lowers the activation energy for emission, but that the pH component has no such effect.

The carotenoid shift in Rhodopseudomonas sphaeroides. The flash induced change.

A mutant, Rhodopseudomonas sphaeroides GIC, having only one major carotenoid, neurosporene, is described. The spectrum of the carotenoid shift in this mutant is analysed and it is concluded that only 7-11% of the pigment is involved under conditions of steady-state illumination and that this pigment undergoes a shift of 7 nm. The spectrum of the carotenoid shift under conditions of multi-flash illumination is examined for changes in shape concordant with a progressive red shift of the pigment with increasing membrane potential; the spectra of the fast change after each of three flashes does not agree well with predictions from a model involving a progressive shift of the pigment, the slow change shows qualitative agreement with such a model but the small size of the signal and the presence of more than one phase makes analysis of this phase more difficult. No separate pool of carotenoid, that might correspond to that postulated to participate in the carotenoid shift, could be identified by fourth derivative analysis of, or curve fitting to, the spectrum of the neurosporene.

The carotenoid shift in Rhodopseudomonas sphaeroides. Change induced under continuous illumination.

The spectrum of the carotenoid shift generated under continuous illumination in the GIC mutant of Rhodopseudomonas sphaeroides, which has a single carotenoid, has been examined under a variety of conditions expected to alter the size of the membrane potential. If the difference spectrum observed was due to a species with the spectrum of the bulk pigment, it would correspond to a change of a variable proportion of the pigment to a form absorbing at a higher wavelength. The maximal change induced by light could be described as a shift of about 10% of the pigment by 7 nm to the red, assuming that the shifted species was spectrally identical to the bulk carotenoid. It is concluded that the changes seen are not easily compatible with a progressive red shift in the whole spectrum with increasing applied potential as would be expected from a simple linear electrochromic mechanism; alternative hypotheses are discussed.

A high potential acceptor for photosystem II.

The effects of ferricyanide on Photosystem II reactions have been investigated by measurements of microsecond and millisecond prompt fluorescence and microsecond-delayed fluorescence in dark-adapted chloroplasts: (1) Titrations using ferri-ferrocyanide mixtures on: (a) the fast phase of the increase in fluorescence yield observed during a xenon flash, and (b) the normalised area above the millisecond fluorescence induction curve for chloroplasts inhibited by DCMU, showed a pH dependent mid point potential of 400 mV at pH 7.0 which varied by approx. -60 mV/pH unit between pH 6 and 8.5. (2) A saturating laser flash induced a fluorescence increase (as monitored by a weak measuring beam) of only 50% of that reached following a second flash in chloroplasts preincubated with ferricyanide and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prior to illumination. In the absence of ferricyanide, the fluorescence level reached after a single flash was initially close to that measured after a second flash (although the level subsequently declined). (3) The initial amplitude of the microsecond-delayed fluorescence excited by a single laser flash was diminished in chloroplasts dark-adapted with ferricyanide. In the presence of DCMU and ferricyanide, the amplitude was also diminished for the first flash of a series, but subsequently enhanced above the level obtained in chloroplasts in the presence of DCMU alone. (4) The above effects were not seen if DCMU was added to the chloroplasts before ferricyanide, or if the period of incubation with ferricyanide was much less than 4 min. (5) These results suggested the presence of a second acceptor Q2, with Em7 = 400 mV and n = 1, before the DCMU block in Photosystem II. There is 0.35--1 equivalent of the acceptor per reaction centre, and its reduction occurs within less than 5 mus. The role of the acceptor in double turnovers of the photochemistry during a single flash and its likely operating redox potential are discussed.

Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.

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

1. In Rhodopseudomonas sphaeroides the Qx absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect. 2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the alpha-band (lambda max at 551--551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c2 (lambda 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, (Formula: see text), 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.

Demonstration of a collisional interaction of ubiquinol with the ubiquinol-cytochrome c2 oxidoreductase complex in chromatophores from Rhodobacter sphaeroides.

Ubiquinone-10 can be extracted from lyophilized chromatophores of Rhodobacter sphaeroides (previously called Rhodopseudomonas sphaeroides) without significant losses in other components of the electron-transfer chain or irreversible damages in the membrane structure. The pool of ubiquinone can be restored with exogenous UQ-10 to sizes larger than the ones in unextracted membranes. The decrease in the pool size has marked effects on the kinetics of reduction of cytochrome b-561 induced by a single flash of light and measured in the presence of antimycin. The initial rate of reduction, which in unextracted preparations increases on reduction of the suspension over the Eh range between 170 and 100 mV (pH 7), is also stimulated in partially UQ-depleted membranes, although at more negative Eh's. When the UQ pool is completely extracted the rate of cytochrome (Cyt) b-561 reduction is low and unaffected by the redox potential. In membranes enriched in UQ-10 above the physiological level the titration curve of the rate of Cyt b-561 reduction is displaced to Eh values more positive than in controls. This effect is saturated when the size of the UQ pool is about 2-3 times larger than the native one. The reduction of Cyt b-561 always occurs a short time after the flash is fired; also the duration of this lag is dependent on Eh and on the size of the UQ pool. A decrease or an increase in the pool size causes a displacement of the titration curve of the lag to more negative or to more positive Eh's, respectively. Similarly, the lag becomes Eh independent and markedly longer than in controls when the pool is completely extracted. These results demonstrate that the rate of turnover of the ubiquinol oxidizing site in the b-c1 complex depends on the actual concentration of ubiquinol present in the membrane and that ubiquinol from the pool is oxidized at this site with a collisional mechanism. Kinetic analysis of the data indicates that this reaction obeys a Michaelis-Menten type equation, with a Km of 3-5 ubiquinol molecules per reaction center.

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 (t 1/2 3-5 microseconds). 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 (102 ms) antimycin-sensitive phase of ferricytochrome c2 reduction, attributed to a specific ubiquinone-10 molecule (Qz), 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 Qz (QzH2) 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 gy 1.90 to gy 1.89, and shifts the Em,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 HII(+); these may reflect additional sites of action of the inhibitor.

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.

Hexane-solubilised reaction centre proteolipid complexes of Rhodopseudomonas sphaeroides.

Reaction centres from Rhodopseudomonas sphaeroides, strain R-26, have been solubilised in hexane by the use of phospholipids and cations. Two procedures have been developed: (I) a two-step technique involving the formation of detergent-free proteoliposomes from detergent solubilised reaction centres and phospholipids and mixing these with hexane in the present of cations; (II) directly sonicating detergent-solubilised reaction centres with phospholipids before mixing with hexane and cations. The yield of the extracted complex varied with the ratios of protein, phospholipid and cations, species of phospholipid, and sonication time. The spectral characteristics of the complex in the organic phase were similar to those of detergent-solubilised reaction centres. The stability of the reaction centres appeared to be dependent on the presence of phospholipid land water in the hexane. The usefulness of the hexane solution as a model membrane system is discussed and its possible future applications are considered.

Asymmetry of an energy transducing membrane the location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata.

Monospecific antibodies have been prepared against cytochrome c2 from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c' from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth. Cytochrome c2 is located in vivo in the periplasmic space between the cell was and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.

Proton-coupled electron transfer at the Q(o) site: what type of mechanism can account for the high activation barrier?

In Rhodobacter sphaeroides, transfer of the first electron in quinol oxidation by the bc(1) complex shows kinetic features (a slow rate (approx. 1.5 x 10(3)/s), high activation energy (approx. 65 kJ/mol) and reorganization energy, lambda (2.5 V)) that are unexpected from Marcus theory and the distances shown by the structures. Reduction of the oxidized iron-sulfur protein occurs after formation of the enzyme-substrate complex, and involves a H-transfer in which the electron transfer occurs through the approx. 7 A of a bridging histidine forming a H-bond with quinol and a ligand to 2Fe-2S. The anomalous kinetic features can be explained by a mechanism in which the electron transfer is constrained by coupled transfer of the proton. We discuss this in the context of mutant strains with modified E(m,7) and pK for the iron-sulfur protein, and Marcus theory for proton-coupled electron transfer. We suggest that transfer of the second proton and electron involve movement of semiquinone in the Q(o) site, and rotation of the Glu of the conserved -PEWY- sequence. Mutational studies show a key role for the domain proximal to heme b(L). The effects of mutation at Tyr-302 (Tyr-279 in bovine sequence) point to a possible linkage between conformational changes in the proximal domain, and changes leading to closure of the iron-sulfur protein access channel at the distal domain.