Cyclic Electron Transfer Research Articles

Characteristics of cyclic electron transport in the cyanobacterium Phormidium laminosum

Cyclic photophosphorylation and ferredoxin-plastoquinone oxidoreductase (FQR) activity have been measured for the first time in the cyanobacterium Phormidium laminosum. They were found to be insensitive to inhibition by up to 10 μM antimycin, as well as to the similarly acting compound, J820. This is in contrast to results from pea thylakoids, but consistent with the other known studies in cyanobacteria and indicates differences between the higher plant and cyanobacterial processes at the level of FQR. The novel inhibitor HQNOBr affected cyclic photophosphorylation at similar concentrations in the two species, but its mode of action differed. In FQR assays with peas, 40 μM HQNOBr had effects consistent with inhibition of the cytochrome bf complex, whereas in P. laminosum the inhibition appeared to be specific to FQR activity. Additional evidence for the specificity of HQNOBr as a cyanobacterial FQR inhibitor comes from the fact that it has no effects on flash-induced absorbance changes at wavelengths characteristics for P700, cytochromes b and f and plastocyanin. This is in contrast to the higher-plant bf complex inhibitor MOA-Stibene which does have effects on flash-induced absorbance changes which are consistent with inhibition of bf complex activity. The data presented indicate that the cyanobacterial and higher-plant cyclic electron transfer processes are similar in over all mechanism but differ in detail, both at the level of FQR activity and the cytochrome bf complex.

The photoinduced cyclic electron transfer in whole cells of Rhodopseudomonas viridis

Light-induced electron transfer has been studied in whole cells of Rhodopseudomonas viridis. No photooxidation of the low-potential hemes of the RC bound cytochrome is observed under the more reducing physiological conditions used in this study, i.e. anaerobiosis. On the other hand, photooxidation of the high-potential heme c 556 of the RC-bound cytochrome is observed 10 μs after flash excitation for cells placed both in aerobic or anaerobic conditions. The photooxidized c 556 is re-reduced by the soluble cytochrome c 2. This electron transfer occurs with an half-time of 110 μs and 40 μs under aerobic and anaerobic conditions, respectively. The half-time of re-reduction of the soluble cytochrome c 2 is also dependent on the redox state of the cells (25 ms and 8 ms under aerobiosis and anaerobiosis, respectively). This re-reduction process is inhibited by addition of 1 mM myxothiazol or stigmatellin, showing that the bc 1 complex is involved in this reaction. Participation of the bc 1 complex in the cyclic electron transfer is also confirmed by the observation of photooxidation of a cytochrome b under anaerobic conditions. Varying the flash intensity or the viscosity of the medium did not affect the half-time of the oxidation of cytochrome c 2. On the other hand, the rate of re-reduction of cytochrome c 2 is strongly affected under such conditions. We conclude from this series of experiments that the soluble cytochrome c 2 forms a stable complex with the RC tetraheme cytochrome. Once oxidized, the cytochrome c 2 diffuses and is re-reduced via a cyclic electron transfer involving the bc 1 complex.

Application of thermostable reaction centers from Chloroflexus aurantiacus as a protonmotive force generating system

Reaction centers (RCs) were purified from the thermophilic phototrophic bacterium Chloroflexus aurantiacus and reconstituted into liposomes. The dependence of cyclic electron transfer via horse-heart cytochrome c, UQ 0 and purified or reconstituted RCs on pH, temperature and ionic strength was investigated. The highest rates of photo-oxidation of cytochrome c were achieved at pH 8 or higher, at 55°C and at an ionic strength below 5 · 10 −4. RCs solubilized with octyl β- d-glucoside could be reconstituted by detergent dialysis into liposomes composed of phospholipids from Escherichia coli or Bacillus stearothermophilus. Upon illumination of RC-containing liposomes in the presence of horse heart cytochrome c and UQ 0 a membrane potential of −160 mV was generated. Maximal values of a membrane potential were generated at 1.1 nmol RC/mg phospholipid. RC-containing liposomes were fused with membrane vesicles from Clostridium fervidus by a freeze/thaw/sonication method (Driessen et al. (1985) Proc. Natl. Acad. Sci. USA 82, 7555–7559). In these hybrid membranes a protonmotive force of −90 mV could be generated upon illumination. The light-induced protonmotive force could drive uptake of l-serine into the hybrid membranes. Incorporation of this thermostable †p- generating system into membrane vesicles from bacteria makes it possible to study secondary transport processes under anaerobic conditions.

Studies on the expression of the pufX polypeptide and its requirement for photoheterotrophic growth in Rhodobacter sphaeroides.

The puf operon in Rhodobacter sphaeroides contains the genes for the light-harvesting antenna complex I (LHI), the reaction centre (RC) L and M subunits and an additional small open reading frame identified as pufX. It has been demonstrated before that a photosynthetically incompetent pufLMX deletion strain was not complemented by a plasmid-borne truncated puf operon version lacking only pufX, although expression of the pufL and pufM gene products was restored. We demonstrate here that the functional reinsertion of only the pufX open reading frame into the same construct is sufficient and necessary for complementation of the non-photosynthetic phenotype. We also demonstrate that the observed lack of photoheterotrophic growth in the absence of pufX is not the result of decreased light-harvesting ability, but rather the result of an impairment in light-driven cyclic electron transfer. Western blots using polyclonal antibodies against a synthetic peptide corresponding to a portion of the DNA-derived pufX amino acid sequence showed that the pufX open reading frame is expressed and that the gene product has an M(r) of 8-10,000 on SDS gels; a value close to the predicted mass of 9 kDa. The pufX polypeptide was localized to the intracytoplasmic membrane fraction and appeared to co-purify with the RC-LHI complex. It is suggested that the pufX polypeptide is associated with the RC-LHI complex and that it may play a critical role in facilitating the interaction between this complex and other components required for light-driven cyclic electron transfer.

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.

Zinc and magnesium substitution in hemoglobin: cyclic electron transfer within mixed-metal hybrids and crystal structure of MgHb

Zinc and magnesium substitution in hemoglobin: cyclic electron transfer within mixed-metal hybrids and crystal structure of MgHb

Electron transfer from cytochrome f to photosystem I in green algae

The time course of P700(+) reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700(+) was reduced with a half-time of 4 μs. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a 'slow-oxidation' state (t 1/2=500 μs) to a 'fast-oxidation' state (t 1/2=80 μs). The addition of ionophore strongly favours and stabilizes the 'fast-oxidation' state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the 'light state' transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.

Dissipation of excitation energy by Photosystem II particles at low pH

The effect of decreasing the pH on the properties of Photosystem II particles isolated from pea and spinach has been investigated. Between pH 6.5 and 4.0 there was a decrease in quantum efficiency of oxygen evolution that was not associated with a proportional closure of reaction centres, as determined by the fluorescence coefficient, qP. This decrease in the quantum yield of Photosystem II appeared to be due to two separate mechanisms. Between pH 6.5 and 5.0, the decrease in quantum yield was not associated with any change in fluorescence yield or in the ratio of Fv/Fm; the decrease was greater at limiting light than in saturating light and may be caused by a cyclic electron transfer that is associated with the destabilisation of the oxidised intermediates of the oxygen-evolving complex and an accelerated rate of Q−A oxidation. Below pH 5.0 the decrease in efficiency is correlated with a quenching of chlorophyll fluorescence. This quenching was light-dependent and required oxidising conditions; redox titrations indicated that a component with a midpoint oxidation reduction potential of +405 mV at pH 4.0 needed to be oxidised if the low pH-dependent quenching was to be observed. The relationship between these effects of low pH and the ‘down-regulation’ of Photosystem II observed under physiological conditions is discussed.

REDOX PROTEINS AS ELECTRON ACCEPTORS FROM CHLOROPHYLL TRIPLET STATE IN LIPID BILAYER VESICLES: KINETICS OF REDUCTION OF MEMBRANE RECONSTITUTED CYTOCHROME c OXIDASE MEDIATED BY 2,5‐DI‐t‐BUTYL BENZOQUINONE AND CYTOCHROME c

Laser flash photolysis was used to determine the kinetics of electron transfer between membrane-bound triplet chlorophyll (3C), cytochrome c (cyt c) located in the external water phase, and vesicle-reconstituted cytochrome c oxidase (CCO). 2,5-Di-t-butyl benzoquinone (2,5 TBQ) was used as an electron transfer mediator between 3C and cyt c. A light-induced cyclic electron transfer sequence between the redox components was observed (3C----2.5 TBQ----cyt c----CCO----C+.). Under optimum conditions of membrane surface charge and ionic strength, the overall efficiency of CCO reduction (based on 3C generated by the laser flash) was 14%. Under the anaerobic conditions used, CCO reoxidation (occurring via electron transfer to C+.) was quite slow (halftime approx. 1 s at 75 mM ionic strength). The multicomponent system displayed a high level of stability, as indicated by its ability to undergo many cycles of reduction and reoxidation without any apparent degradation of the components. These results demonstrate the feasibility of constructing complex electron transfer chains, including both soluble and membrane-bound redox proteins, in artificial lipid bilayers, whose properties can be readily controlled by manipulating parameters such as ionic strength and membrane composition.

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus

1. The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone-10 two multiprotein complexes: the reaction center and the ubiquinol-cytochrome-c2 reductase (or bc1 complex). 2. In the presence of cytochrome c2 added externally, at concentrations in the range 10-10(4) nM, a flash-induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone-reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo-oxidized reaction center. At low ionic strength (10 mM KCl) and at concentrations of cytochrome c2 lower than 1 microM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half-time for cytochrome b561 reduction is about 3 ms. 3. At high ionic stength (200 mM KCl), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome-b561 reduction. If the absolute values of these rates are compared with those of the cytochrome-c2-reaction-center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate-limiting and involves electrstatic interactions. 4. A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second-order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second-order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 X 10(-11) cm2 s-1 can be evaluated.

Binding-protein-dependent alanine transport in Rhodobacter sphaeroides is regulated by the internal pH.

The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.

Functional reconstitution of photosynthetic cyclic electron transfer in liposomes

The interaction of solubilized reaction centers from the phototrophic bacterium, Rhodobacter sphaeroides , and the solubilized ubiquinol-cytochrome c oxidoreductase of Rhodobacter capsulatus was studied in solution and after coreconstitution in liposomes prepared from Escherichia coli phospholipids. Under both conditions, the ubiquinol-cytochrome c oxidoreductase increased the light-induced cyclic electron transfer, induced by reaction centers with 2,3-dimethoxy-5-methyl-6-(prenyl) 2 -1,4-benzoqu and cytochrome c as redox mediators. This effect was more pronounced at acid pH values. The light-induced cyclic electron transfer in these liposomes resulted in the generation of a protonmotive force. Under conditions where the protonmotive force was composed of a membrane potential only, the highest membrane potential (approx. −200 mV) was generated when 2,3-dimethoxy-5-methyl-6-(prenyl) 10 -1,4-benzo-quinone was used as redox mediator and when both electron transfer proteins were co-reconstituted in a 2:1 molar ratio. At acid pH non-transitent membrane potentials could be generated only in liposomes containing both reaction centers and the ubiquinol-cytochrome c oxidoreductase. These observations show that the pH-dependent direct oxidation of cytochrome c by ubiquinol in the liposomes was indeed catalyzed by the ubiquinol-cytochrome c oxidoreductase and that this oxidoreductase participates in proton pumping. This could also be concluded from the stimulating effect of 2,3-dimethoxy-5-methyl-6-(prenyl) 10 -1,4-benzoquinone on the membrane-potential-generating capacities in liposomes containing both electron transfer complexes. Such a stimulation was not observed in liposomes containing only reaction centers. The presence of cytochrome c in the co-reconstituted system was found to be essential for proton pumping.

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — characteristics and reinterpretation of single-turnover cyclic electron transfer

The contributions of ferredoxin, P-700, plastocyanin and the cytochromes c-554, and b-563 to single-turnover electron transfer in Photosystem (PS) I-enriched subchloroplast vesicles were deconvoluted by fitting the literature-derived spectra of these components to the observed absorption data at a series of wavelengths, according to a linear least-squares method. The obtained corresponding residuals showed that the applied component spectra were satisfactory. The deconvoluted signals of cytochromes c-554 and b-563 differed in some cases significantly from the classical dual-wavelength signals recording at 554–545 nm and 563–575 (or −572) nm, due to interference from other electron-transferring components. KCN, DNP-INT (2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether), DBMIB (2,5-dibromo-3-methyl-6-isopropyl- p-benzo-quinone) and antimycin A all inhibited electron transfer, although antimycin and DBMIB inhibited only after a few turnovers of the cytochrome bf complex. Fast flash-induced reduction of cytochrome b-563 exclusively reflected oxidant-induced reduction. Fast electron flow from cytochrome c-554 to plastocyanin and P-700 resulted in an apparent rereduction of cytochrome c-554 that was slower than the reduction of cytochrome b-563. Model simulations indicate that under highly oxidizing conditions for the Rieske FeS centre and reducing conditions for cytochrome b-563, the semiquinone at the Q z site cannot only reduce cytochrome b-563, but can also oxidize cytochrome b-563 and reduce the Rieske FeS centre. The effect of 10 μM gramicidin D was evaluated in order to determine the contributions by electrochromic absorption changes around 518 nm. Gramicidin left electron transfer, monitored in the 550–600 nm range, unchanged. The gramicidin-sensitive (membrane potential-associated) signal at 518 nm differed from the signals recorded in the absence of gramicidin at 518 nm or 518–545 nm, due to spectral interference from electron-transferring components in the latter signals. KCN, DBMIB and antimycin A affected both the fast and slow components of the electrochromic signal, but did not proportionally affect the initial electron transfer from P-700 to ferredoxin (charge separation in PS I). Not only the slow (10–100 ms) component of the 518 nm absorption change, but also part of the fast (less than 1 ms) component appears to minitor electrogenic events in the cytochrome bf complex.

Cytochrome b-559 may function to protect photosystem II from photoinhibition.

Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.

Structure and function of the chloroplast cytochrome bf complex

The chloroplast cytochrome bf complex is an intrinsic multisubunit protein from the thylakoid membrane consisting of four polypeptides: cytochrome f, a two heme containing cytochrome b 6, the Rieske iron-sulfur protein, and a 17 kD polypeptide of undefined function. The complex functions in electron transfer between PSII and PSI, where most mechanisms suggest that the transfer of a single reducing equivalent from plastoquinol to plastocyanin results in the translocation of two protons across the membrane. Primary sequence analyses, dichroism studies, and functional considerations allow the construction of an approximate structural model of a monomeric complex, although some evidence exists for a dimeric structure. Resolution of the properties of the two cytochrome b 6 hemes has relied upon the availability of purified solubilized complex, while evidence in the thylakoid suggests the difference between the two hemes are not as great in situ. Such variability in the spectroscopic and electrochemical properties of the cytochrome b 6 is a major concern during the experimental use of the purified complex. There is a general consensus that the complex contains a plastoquinol oxidizing (Qz) site, although the evidence for a plastoquinone reduction (Qc) site, called for in most mechanistic hypotheses, is less substantive. Probably the most severe challenge to the so called Q-cycle mechanism comes from experimental observations made with cytochrome b 6 initially reduced, where proposed interpretations more closely resemble a b-cycle than a Q-cycle. Although functional during cyclic electron transfer, the role of the complex and its possible interaction with other proteins, has not been completely resolved.

Protonmotive Q cycle pathway of electron transfer and energy transduction in the three-subunit ubiquinol-cytochrome c oxidoreductase complex of Paracoccus denitrificans.

Ubiquinol-cytochrome c oxidoreductase (cytochrome bc1) complex from Paracoccus denitrificans consists of only three polypeptide subunits (Yang, X., and Trumpower, B. L. (1986) J. Biol. Chem. 261, 12282-12289), whereas the analogous complexes of eukaryotic mitochondria consist of nine or more polypeptides (Schagger, H., Link, T. A., Engel, W. D., and von Jagow, G. (1986) Methods Enzymol. 126, 224-237). Using the purified three-subunit Paracoccus complex we have tested whether this simple cytochrome bc1 complex has the same electron transfer pathway and proton translocation activity as the bc1 complexes of mitochondria. Under presteady state conditions, the effects of inhibitors on reduction of cytochromes b and c1 by quinol and oxidant-induced reduction of cytochrome b indicate a cyclic electron transfer pathway and two routes of cytochrome b reduction in the three-subunit Paracoccus cytochrome bc1 complex. A novel method was developed to incorporate the cytochrome bc1 complex into liposomes with the detergent dodecyl maltoside. The enzyme reconstituted into liposomes translocated protons with an H+/2e value of 3.9. Carbonyl cyanide m-chlorophenylhydrazone eliminated proton translocation, while permitting the scalar release of protons from quinol, and thus reduced the H+/2e ratio to 2. These values agree with the predicted stoichiometries for proton translocation by a protonmotive Q cycle pathway. No inhibition of proton translocation by N',N'-dicyclohexylcarbodiimide was detected when the Paracoccus cytochrome bc1 complex was incubated with N',N'-dicyclohexylcarbodiimide before or after reconstitution into liposomes. Electron transfer in the three-subunit complex thus appears to occur by a protonmotive Q cycle pathway identical to that in mitochondrial cytochrome bc1 complexes. Only three polypeptides, cytochromes b, c1, and the Rieske iron-sulfur protein, are required for respiration and energy transduction in the cytochrome bc1 complex. The function of the supernumerary polypeptides in mitochondrial bc1 complexes is thus unclear.

Electron Donation in Photosystem II

AbstractThe pathways of electron donation in Photosystem II (PS II) as studied by electron paramagnetic resonance (EPR) and time‐resolved optical spectroscopy are discussed. The EPR studies have defined two competing pathways of electron donation in PS II, from cytochrome b559 and from the Mn site of the oxygen‐evolving center. The kinetics of re‐reduction of the primary electron donor of PS II (P680). as measured by optical spectroscopy, are re‐evaluated in light of the EPR results. We propose that the 35‐μs kinetic component is due to the reduction of chlorophyll, an alternate electron donor on the cytochrome b559 pathway, rather than to the reduction of P680. The chlorophyll/cytochrome b559 pathway has been proposed to be part of a cyclic electron transfer pathway around PS II; we suggest that photooxidation of chlorophyll is the first step leading to photoinhibition and that cytochrome b559 serves to protect PS II from photoinhibition by rapidly re‐reducing the oxidized chlorophyll (Thompson, L.K.; Brudvig, G.W. Biochemistry, 1988, 27: 6653). These results and proposals are summarized in an overall scheme of electron transfer pathways and rates in PS II.

Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus

The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c 1 and b of the cytochrome bc 1 complex, alone or in combination with deletion of the gene for cytochrome c 2. Deletion of cytochrome c 1 renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c 2, because in the absence of the bc 1 complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c 2 is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc 1 complex. The deletion of cytochromes c 1 and c 2 reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with E m7 = + 312 mV and E m6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.

Reaction centers from Rhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation.

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochrome c and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ0), a lipid- and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ0H2) and oxidized cytochrome c. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochrome c and UQ0. At 250 and 25 microM UQ0 and cytochrome c, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O(2) (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP(+) ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.