Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of three cytochromes in photosynthesis of whole cells of Rhodospirillum rubrum as studied by flash spectroscopy. Evidence for two types of reaction center.

1. Changes in the absorption spectrum induced by 10-mus flashes and continuous light of various intensities were studied in whole cells of Rhodospirillum rubrum in the presence and absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide(HOQNO) and antimycin A. 2. Three cytochromes, c-420 (cytochrome c2), c-560 (cytochrome b) and c-428 were photoactive and gamma and alpha peaks at 420 and 550, 428 and 560, and 428 and 551 nm, respectively; they were photooxidized following the flash with half times of 0.3, 0.6 and 7 ms in the approximate ratios of 1/100, 1/300 and 1/1000 (cytochrome oxidized/antenna chlorophyll) and became reduced with half times of 12 ms, 60 ms and 0.7 s, respectively. c-428 and c-560 have not been distinguished before. 3. From a detailed analysis of the kinetics of P+ (oxidized reaction center chlorophyll) and the cytochromes, we conclude that 5% of the P+ (P2+) oxidizes c-428, whereas the remaining 95% of P+ (P1+) oxidizes c-420. At actinic light intensities low enough to keep c-420 fully reduced, approx. 4-5% of P becomes oxidized, accompanied by all c-428. The P2+ -P2 difference spectrum induced by this weak light is, when corrected for a shift to longer wavelengths of the bacteriochlorophyll absorption band at 878 nm, identical to the difference spectrum caused by the photooxidation of the remaining P1. At low flash intensity, c-428 becomes preferentially photooxidized, which suggests that the reaction centers where c-428 functions as a secondary donor contain much more antenna pigments compared to the centers where c-420 serves this purpose. 4. c+-420 is reduced in a competitive way by reduced c-560 (t 1/2=7 ms), and by an electron donor pool, (t 1/2=15 ms). HOQNO inhibits both pathways; antimycin A only the first. In the presence of HOQNO, c-560 is in the oxidized state in the dark, and is reduced in a light flash (t 1/2=100 ms), indicating that c-560 acts in a cyclic electron transport chain connected to P1. 5. The ratio of numbers of molecules P1 and antenna bacteriochlorophyll, transferring excitation energy to P1, is P1/bacteriochlorophyll1=1/30 P2: bacteriochlorophyll2=1/300; c-420/P1=1:2; c-560/P1=1/6; C-428/P2=1/1; bacteriochlorophyll2=7:3. If P2 is oxidized, excitation energy is transferred from bacteriochlorophyll2 to bacteriochlorophyll1.     

The role of high-potential iron protein and cytochrome c(8) as alternative electron donors to the reaction center of Chromatium vinosum

Under anaerobic conditions, intact cells of the purple sulfur bacterium Chromatium vinosum exhibit rapid photooxidation of the two low-potential hemes of the c-type cytochrome associated with the reaction center, after exposure to two short light flashes separated by a dark interval. Reduction of the photooxidized low-potential hemes is very slow under these conditions. On subsequent flashes, rapid photooxidation of a high-potential reaction center heme occurs and is followed by its rereduction on the millisecond time scale. Cells maintained under aerobic conditions exhibit the millisecond time scale reduction of the photooxidized high-potential heme after each flash. Cells grown autotrophically in the presence of Na(2)S and Na(2)S(2)O(3) appear to use the soluble [4Fe-4S]-containing protein, HiPIP, as the only direct electron donor to the reaction center heme under aerobic conditions. In contrast, cells grown in the presence of organic compounds, but in the absence of Na(2)S and Na(2)S(2)O(3), appear to use a soluble c-type cytochrome (most likely cytochrome c(8)) as the only electron donor to the reaction center heme under aerobic conditions. Cells grown autotrophically, in the presence of Na(2)S and Na(2)S(2)O(3), have a slightly higher ratio of HiPIP to cytochrome c(8) and a ratio of Rieske iron-sulfur protein to reaction center that is approximately one-half that of cells grown in the absence of Na(2)S and Na(2)S(2)O(3) but in the presence of organic compounds.     

Electron transfer kinetics in purified reaction centers from the green sulfur bacterium Chlorobium tepidum studied by multiple-flash excitation.

Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the secondary electron acceptor, QB, has been characterized by light-induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB- state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB-. Assignments are proposed for the C...O vibration of QB- and tentatively for the C = O and C = C vibrations of QB.     

Laser-induced reactions of P700 and cytochrome f in a blue-green alga, Plectonema boryanum

Kinetics of the absorption change of P700 (blue band) and cytochrome f in whole cells of a blue-green alga, Plectonema boryanum, have been studied by Q-switched ruby-laser flash excitation (694 nm; approx. 20 nsec) to elucidate the sequential relationship of these two components in photosynthetic electron transport. “P700” was photooxidized within 2 μsec and recovered in two phases t_1/2 10 μsec and 200 μsec). Under the same conditions cytochrome f was oxidized with a half time of 15 μsec. The magnitude of the fast phase of “P700” recovery, however, diminished at lower laser intensity while the cytochrome f change remained unaffected. The result suggests that cytochrome f and P700 may not be on the same electron-transport chain.     

Kinetics of reduction of the oxidized primary electron donor of photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (deltaA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures. In the microsecond time range the difference spectrum of deltaA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P+-700; it decays in a polyphasic manner with half-times of 17 microseconds, 210 microseconds and over 1 ms. The oxidized primary donor of Photosystem II (P+II) is not detected with a time resolution of 3 microseconds. After treatment with 3--10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P+II is observed and decays biphasically (a major phase with t1/2=20--40 microseconds, and a minor phase with t1/2 congruent to 200 microseconds), probably by reduction by an accessory electron donor. In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P+II is reduced with a half-time of 25--45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Steady-state cyclic electron transfer through solubilized Rhodobacter sphaeroides reaction centres

The mechanism, thermodynamics and kinetics of light-induced cyclic electron transfer have been studied in a model energy-transducing system consisting of solubilized Rhodobacter sphaeroides reaction center/light harvesting-1 complexes (so-called core complexes), horse heart cytochrome c and a ubiquinone-0/ubiquinol-0 pool. An analysis of the steady-state kinetics of cytochrome c reduction by ubiquinol-0, after a light-induced steady-state electron flow had been attained, showed that the rate of this reaction is primarily controlled by the one-electron oxidation of the ubiquinol-anion. Re-reduction of the light-oxidized reaction center primary donor by cytochrome c was measured at different reduction levels of the ubiquinone-0/ubiquinol-0 pool. These experiments involved single turnover flash excitation on top of background illumination that elicited steady-state cyclic electron transfer. At low reduction levels of the ubiquinone-0/ubiquinol-0 pool, the total cytochrome c concentration had a major control over the rate of reduction of the primary donor. This control was lost at higher reduction levels of the ubiquinone/ubiquinol-pool, and possible reasons for this behaviour are discussed.     

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroids: the physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions.

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c2 in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferricytochrome with a halftime of 1-2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction. The state of reduction of Z, which may be a quinone.protein complex near the inner (cytochrome c2) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c2, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome rereduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c2 oxido-reductase.     

Recombination reduction of photooxidized cytochrome c in reaction centers of Rhodopseudomonas viridis at low temperature

The temperature dependence of dark reduction of photooxidized cytochrome c was studied in isolated preparations of Rhodopseudomonas viridis reaction centers. Within the range from room temperature to 260 K this process was found to be mediated by thermal diffusion of exogenous donor molecules, whereas at lower temperatures photooxidized cytochrome is reduced as a result of indirect recombination with photoreduced primary quinone acceptor. Kinetic simulation allowed certain thermodynamic characteristics of this reaction to be calculated. To the first approximation, these characteristics correlate with the estimates obtained from the results of direct redox titration.     

The role of c-type cytochromes in the photosynthetic electron transport pathway of Rhodobacter capsulatus

(1) Short flash excitation of membrane vesicles of a cytochrome-c2-deficient mutant of Rhodobacter capsulatus (strain MT-G4/S4) led to rapid oxidation of a c-type cytochrome. In redox titrations, the photooxidation of c-type cytochrome was attenuated with a midpoint of approx. +360 mV. Vesicles from a control strain, MT1131, gave similar results. These findings are consistent with those of Prince et al. (Prince, R.C., Davidson, E., Haith, L.E. and Daldal, F. (1986) Biochemistry 25, 5208-5214). (2) In anaerobic intact cells the extent of rapid re-reduction of c-type cytochrome oxidised after a flash was less in MT-G/S4 than in MT1131. Cytochrome c reduction in both strains was inhibited by myxothiazol. The myxothiazol-sensitive component of the electrochromic absorbance change in cells indicated that rapid charge separation through the cytochrome bc1 complex was less extensive after a flash in MT-G4/S4 than in MT 1131. (3) In anaerobic intact cells and in chromatophores of Rb. capsulatus strain MT-GS18, a mutant deficient in both cytochrome c1 and cytochrome c2, flash excitation led to the oxidation of c-type cytochrome. Redox titrations and spectra of chromatophores suggested that this is the same cytochrome as was photooxidized in vesicles of MT-G4/S4 and MT1131. This result is in contrast with earlier findings (Prince, R.C. and Daldal, F. (1987) Biochim. Biophys, Acta 894, 370-378) in which it was reported that no photooxidation of c-type cytochrome occurred in the absence of c1 and c2, and argues against the possibility that cytochrome c1 can rapidly and directly donate electrons to the reaction centre. (4) It is proposed that a previously uncharacterized, membrane-bound c-type cytochrome (Em7 approximately +360 mV) is present in Rb-capsulatus MT1131, in the c2-deficient mutant MT-G4/34 and in the c1/c2-deficient mutant MTGS18. This cytochrome and cytochrome c2 are alternative electron donors to the reaction centre in strain MT1131.     

Photo-induced electron transfer in intact cells of Rubrivivax gelatinosus mutants deleted in the RC-bound tetraheme cytochrome: Insight into evolution of photosynthetic electron transport

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc(1) complex is the High Potential Iron-sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron-sulfur Protein, this connection is achieved by the high potential cytochrome c(8). Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P(+) and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.     

Photosynthetic membrane development in Rhodopseudomonas sphaeroides. Spectral and kinetic characterization of redox components of light-driven electron flow in apparent photosynthetic membrane growth initiation sites

The kinetics of light-driven electron flow and the nature of redox centers at apparent photosynthetic membrane growth initiation sites in Rhodopseudomans sphaeroides were compared to those of intracytoplasmic photosynthetic membranes. In sucrose gradients, these membrane growth sites sediment more slowly than intracytoplasmic membrane-derived chromatophores and form an upper pigmented band. Cytochromes c1, c2, b561, and b566 were demonstrated in the upper fraction by redox potentiometry; c-type cytochromes were also detected electrophoretically. Signals characteristic of light-induced reaction center bacteriochlorophyll triplet and photooxidized reaction center bacteriochlorophyll dimer states were observed by EPR spectroscopy but the Rieske iron-sulfur signal of the ubiquinol-cytochrome c2 oxidoreductase was present at a 3-fold reduced level on a reaction center basis in comparison to chromatophores. Flash-induced absorbance measurements of the upper pigmented fraction demonstrated reaction center primary and secondary semiquinone anion acceptor signals, but cytochrome b561 photoreduction and cytochrome c1/c2 reactions occurred at slow rates. This fraction was enriched approximately 2- and 4-fold in total b- and c-type cytochromes, respectively, per reaction center over chromatophores, but photoreducible b-type cytochrome was lower. Measurements of respiratory activity indicated a 1.6-fold higher level of succinate-cytochrome c oxidoreductase/reaction center than in chromatophores, but the apparent turnover rates in both preparations were low. Overall, the results suggest that complete cycles of rapid, light-driven electron flow do not occur merely by introduction of newly synthesized reaction centers into respiratory membrane, but that subsequent synthesis and assembly of appropriate components of the ubiquinol-cytochrome c2 oxidoreductase is required.     

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients.

Two independent sets of measurements using a variety of methods show that a sample with A_{800 nm} = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1.

Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77.

These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: _{802 nm} = 288 (± 14) mM⁻¹ · cm⁻¹ and _{865 nm} = 128 (± 6) mM⁻¹ · cm⁻¹. For reversible bleaching of the 865-nm band, Δ^{red - ox}_865nm = 112 (± 6) mM⁻¹ · cm⁻¹ (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.     

Kinetic properties of the acceptor quinone complex in Rhodopseudomonas viridis

The kinetics of electron transfer from the primary (QA) to the secondary (QB) quinone acceptor in whole cells and chromatophores of Rhodopseudomonas viridis was studied as a function of the redox state of QB and of pH by using a photovoltage technique. Under conditions where QB was oxidized, the reoxidation of QA- was found to be essentially monophasic and independent of pH with a half-time of about 20 microseconds. When QB was reduced to the semiquinone form by a preflash, the reoxidation of QA- was slowed down showing a half-time between 40 and 80 microseconds at pH less than or equal to 9. Above pH 9, the rate of the second electron transfer decreased nearly one order of magnitude per pH unit. After a further preflash, the fast and pH-independent kinetics of QA- reoxidation was essentially restored. The concentration of QA still reduced 100 microseconds after its complete reduction by a flash showed distinct binary oscillations as a function of the number of preflashes, confirming the interpretation that the electron-transfer rate depends on the redox state of QB. After addition of o-phenanthroline, the reoxidation of QA- is slowed down to the time range of seconds as expected for a back-reaction with oxidized cytochrome. Under conditions where inhibitors of the electron transfer between the quinones fail to block this reaction in a fraction of the reaction centers due to the presence of the extremely stable and strongly bound semiquinone, QB-, these reaction centers show a slow electron transfer on the first flash and a fast one on the second, i.e., an out-of-phase oscillation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: II. Interaction with external Electron Donors and Acceptors and a Reevaluation of Some Spectroscopic Data

Photochemical reaction centers prepared from Rhodopseudomonas spheroides were treated with reduced cytochrome c (cyt c), and in some cases with ubiquinone (UQ), and illuminated. The light-induced oxidation of cy and reduction of UQ were observed, and also the variations in fluorescence of P870. These observations indicated that each reaction center contains a primary photochemical electron acceptor capable of holding just one electron. Depending on the method of preparation, the reaction centers may also contain secondary electron acceptor pools consisting mainly of UQ. The role of native UQ as an electron acceptor could be duplicated by added UQ. The yield of P870 fluorescence increased by a factor of 3-4, at most, during illumination of reaction centers in the presence of an electron donor such as reduced cyt. This suggests that the quantum efficiency for the primary photoact is about 0.7, rather than 0.9-1.0 as concluded in the past from optical absorption measurements. The apparent quantum efficiency for the oxidation of cyt by illuminated reaction centers can be increased by the addition of UQ and is decreased at higher concentrations of the detergent lauryl dimethylamine oxide (LDAO). These treatments do not affect the quantum efficiency of P870 oxidation, measured in the absence of cyt.     

Discrete catalytic sites for quinone in the ubiquinol-cytochrome c2 oxidoreductase of Rhodopseudomonas capsulata. Evidence from a mutant defective in ubiquinol oxidation

A non-photosynthetic mutant (Ps-) of Rhodopseudomonas capsulata, designated R126, was analyzed for a defect in the cyclic electron transfer system. Compared to a Ps+ strain MR126, the mutant was shown to have a full complement of electron transfer components (reaction centers, ubiquinone-10, cytochromes b, c1, and c2, the Rieske 2-iron, 2-sulfur (Rieske FeS) center, and the antimycin-sensitive semiquinone). Functionally, mutant R126 failed to catalyze complete cytochrome c1 + c2 re-reduction or cytochrome b reduction following a short (10 microseconds) flash of actinic light. Evidence (from flash-induced carotenoid band shift) was characteristic of inhibition of electron transfer proximal to cytochrome c1 of the ubiquinol-cytochrome c2 oxidoreductase. Three lines of evidence indicate that the lesion of R126 disrupts electron transfer from quinol to Rieske FeS: 1) the degree of cytochrome c1 + c2 re-reduction following a flash is indicative of electron transfer from Rieske FeS to cytochrome c1 + c2 without redox equilibration with an additional electron from a quinol; 2) inhibitors that act at the Qz site and raise the Rieske FeS midpoint redox potential (Em), namely 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole or 3-alkyl-2-hydroxy-1,4-napthoquinone, have no effect on cytochrome c1 + c2 oxidation in R126; 3) the Rieske FeS center, although it exhibits normal redox behavior, is unable to report the redox state of the quinone pool, as metered by its EPR line shape properties. Flash-induced proton binding in R126 is indicative of normal functional primary (QA) and secondary (QB) electron acceptor activity of the photosynthetic reaction center. The Qc functional site of cytochrome bc1 is intact in R126 as measured by the existence of antimycin-sensitive, flash-induced cytochrome b reduction.     

Single and multiple turnover reactions in the ubiquinone-cytochrome b-c2 oxidoreductase of Rhodopseudomonas sphaeroides. The physical chemistry of the major electron donor to cytochrome c2, and its coupled reactions

We have examined the thermodynamic properties of the physiological electron donor to ferricytochrome c₂ in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides. This donor (Z), which is capable of reducing the ferri-cytochrome with a halftime of 1–2 ms under optimal conditions, has an oxidation-reduction midpoint potential of close to 150 mV at pH 7.0, and apparently requires two electrons and two protons for its equilibrium reduction.

The state of reduction of Z, which may be a quinone · protein complex near the inner (cytochrome c₂) side of the membrane, appears to govern the rate at which the cyclic photosynthetic electron transport system can operate. If Z is oxidized prior to the flash-oxidation of cytochrome c₂, the re-reduction of the cytochrome takes hundreds of milliseconds and no third phase of the carotenoid bandshift occurs. In contrast if Z is reduced before flash activation, the cytochrome is rereduced within milliseconds and the third phase of the carotenoid bandshift occurs. The prior reduction of Z also has a dramatic effect on the uncoupler sensitivity of the rate of electron flow; if it is oxidized prior to activation, uncoupler can stimulate the cytochrome re-reduction after several turnovers by less than tenfold, but if it is reduced prior to activation, the stimulation after several turnovers can be as dramatic as a thousandfold. The results suggest that Z plays a central role in controlling electron and proton movements in the ubiquinone cytochrome b-c₂ oxido-reductase.