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P Joliot

Publications and source records attributed to P Joliot.

At least 19 recordsLinked to original sources
PubMed2001

Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c(6) in Synechococcus sp. PCC 7002 in vivo.

Cytochrome c(6) donates electrons to photosystem I (PS I) in Synechococcus sp. PCC 7002. In this work, we provide evidence for rapid electron transfer (t(1/2) = 3 micros) from cytochrome c(6) to PS I in this cyanobacterium in vivo, indicating prefixation of the reduced donor protein to the photosystem. We have investigated the cytochrome c(6)-PS I interaction by laser flash-induced spectroscopy of intact and broken cells and by redox titrations of membrane and supernatant fractions. Redox studies revealed the expected membrane-bound cytochrome f, b(6), and b(559) species and two soluble cytochromes with alpha-band absorption peaks of 551 and 553 nm and midpoint potentials of -100 and 370 mV, respectively. The characteristics and the symmetrical alpha-band spectrum of the latter correspond to typical cyanobacterial cytochrome c(6) proteins. Rapid oxidation of cytochrome c(6) by PS I in vivo results in a unique, asymmetric oxidation spectrum, which differs significantly from the spectra obtained for cytochrome c(6) in solution. The basis for the unusual cytochrome c(6) spectrum and possible mechanisms of cytochrome c(6) fixation to PS I are discussed. The occurrence of rapid electron transfer to PS I in cyanobacteria suggests that this mechanism evolved before the endosymbiotic origin of chloroplasts. Its selective advantage may lie in protection against photo-oxidative damage as shown for Chlamydomonas.

Cyanobacteria↗
PubMed2001

Evidence for two active branches for electron transfer in photosystem I.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

Amino Acid Sequence↗
PubMed2001

Electrogenic events associated with electron and proton transfers within the cytochrome b(6)/f complex.

The kinetics and amplitude of the membrane potential changes associated with electron and proton transfers within the cytochrome b(6)/f (cyt b/f) complex (phase b) are measured in vivo in Chlamydomonas reinhardtii under anaerobic conditions. Upon saturating flash excitation, fast components in the membrane potential decay superimposed on phase b lead to an underestimation of the amplitude of this phase. In the FUD50 mutant strain, which lacks the ATP synthase, the decay of the membrane potential is slowed down compared to the wild type, and the kinetics and amplitude of phase b may be accurately determined. This amplitude corresponds to the transfer of at least 1.5 charges across the membrane per positive charge transferred to photosystem I, whatever the flash energy. This value largely exceeds that predicted by a Q-cycle process. Similar conclusions are reached using the wild type strain in the presence of 9 microM dicyclohexylcarbodiimide, which specifically inhibits the ATP synthase. It is concluded that a proton pumping process is operating in parallel with the Q-cycle, with a yield of approximately 0.5 proton pumped by cyt b/f complex turnover, irrespective of the flash energy.

Adenosine Triphosphatases↗
PubMed1999

In vivo analysis of the electron transfer within photosystem I: are the two phylloquinones involved?

Electron transfer within PS I reaction centers has been analyzed in vivo in a mutant of Chlorella sorokiniana which lacks most of the PS II and of the peripheric antennae, using a new spectrophotometric technique with a time resolution of approximately 5 ns. Absorption changes associated with the oxidation of semiphylloquinone (acceptor A(1)(-)) have been characterized in the 371-545 nm spectral range. The oxidation of A(1)(-) and the reduction of an iron-sulfur cluster (F(X), F(A)F(B)) is monitored by an absorption decrease at 377 nm (semiphylloquinone absorption band) and by the decrease of two positive absorption bands around 480 and 515 nm, respectively, very likely associated with a local electrochromic shift induced by A(1)(-) on a carotenoid molecule localized in its vicinity. A(1)(-) undergoes a two-phase oxidation of about equal amplitude with half-times of approximately 18 and approximately 160 ns, respectively. Two hypotheses are proposed to interpret these data: (1) Photosystem I reaction centers are present under two conformational states which differ by the reoxidation rate of A(1)(-). (2) The two phylloquinones corresponding to the two branches of the PS I heterodimer are involved in the electron transfer. The similar amplitude of the two phases implies that the rates of electron transfer from P700 to each of the phylloquinones are about equal. The two different rate constants measured for A(1)(-) oxidation suggests some asymmetry in the relative position of the two phylloquinones with respect to F(X).

Carotenoids↗
PubMed1999

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.

Electron Transport Complex III↗
PubMed1999

The photosynthetic apparatus of Rhodobacter sphaeroides.

Functional and ultrastructural studies have indicated that the components of the photosynthetic apparatus of Rhodobacter sphaeroides are highly organized. This organization favors rapid electron transfer that is unimpeded by reactant diffusion. The light-harvesting complexes only partially surround the photochemical reaction center, which ensures an efficient shuttling of quinones between the photochemical reaction center and the bc1 complex.

Electron Transport↗
PubMed1998

Glu78, from the conserved PEWY sequence of subunit IV, has a key function in cytochrome b6f turnover.

We have investigated the structure to function relationship at the Qo site in cytochrome b6f complexes in vivo. To this end, we created site-directed mutants of Chlamydomonas reinhardtii, at position 78 in the sequence of subunit IV. The target glutamic acid, present in the highly conserved 77PEWY80 sequence, was changed to residues of different polarities which did not prevent the functional assembly of cytochrome b6f complexes. Spectroscopic analysis performed in anaerobic conditions in vivo revealed distinct alterations in cytochrome b6f function, depending on the nature of the substituted residue. The semiconservative E78D substitution, in which only the length of the side chain is reduced, retained the functional features of the wild-type configuration. The E78K and E78L substitutions caused a significant decrease, by factors of 3 and 5, respectively, in the rate of the concerted oxidation process at the Qo site without a change in the affinity of Qo for reduced plastoquinones. The E78Q and E78N substitutions modified the characteristics of cytochrome b6f turnover under repetitive flash illumination. They caused a large increase in the electrogenicity of the electron-transfer reactions through the mutated cytochrome b6f complex. This increase was specifically sensitive to the electrical component of the proton-motive force. Surprisingly, despite the larger number of charges translocated across the membrane per charge injected in the high potential chain, the reduction phase for cytochrome b6 became barely detectable in the mutants, unless inhibitors at the Qi site were present. We show that similar functional characteristics can be observed with the cytochrome b6f complex in the wild-type in anaerobic conditions, provided a single flash illumination regime is used. These observations suggest that cytochrome b6f turnover may involve a mechanism implying an extra proton pumping activity.

Amino Acid Sequence↗
PubMed1998

In vivo analysis of the effect of dicyclohexylcarbodiimide on electron and proton transfers in cytochrome bf complex of Chlorella sorokiniana.

The effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron and proton transfers within the cytochrome (cyt) bf complex has been analyzed in living cells of the green algae Chlorella sorokiniana under anaerobic conditions. DCCD induces a partial decoupling of the protomotive Q-cycle, in agreement with the conclusions of Wang and Beattie (1991) Arch. Biochem. Biophys. 291, 363-370. In the presence of 20 microM DCCD, we observe the development of a lag phase in the kinetics of the slow electrogenic phase associated with electron and proton transfers within the cyt bf complex. In the same conditions, the initial rate of cyt b and cyt f reduction is decreased by about 30%. We propose that in the absence of DCCD, a transmembrane movement of proton is coupled to the oxidation of plastoquinol at site Qo. In the presence of 20 microM DCCD, this redox-coupled proton pump is inhibited, and the kinetics of phase b and cyt b reduction become close to that predicted on the basis of a pure Q-cycle process. In agreement with this hypothesis, we observe that upon a weak-flash excitation, two charges are translocated through the membrane in addition to the charge translocated at the level of photosystem I. Part of this large electrogenic phase could be associated with the translocation of a proton from the stroma to the lumen. A tentative mechanism is discussed that remains in the frame of the Q-cycle but accounts for an additional proton-pumping process or for the partial decoupling observed in the presence of DCCD, as well.

Anaerobiosis↗
PubMed1997

Electron transfer towards the RCI-type photosystem in the green sulphur bacterium Chlorobium limicola forma thiosulphatophilum studied by time-resolved optical spectroscopy in vivo.

Flash-induced spectral changes in the wavelength region of the alpha-peaks of heme proteins and in the time domain from microseconds to seconds have been recorded on whole cells of the green sulphur bacterium Chlorobium limicola forma thiosulfatophilum. Extensive flash-excitation by trains of flashes resulted in oxidation of 7-8 c-type heme molecules/photosynthetic reaction centre. The complement of heme species was found to be spectrally heterogeneous allowing the study of electron transfer events induced by an isolated single-turnover flash. Under single-flash conditions, a c553 heme was seen to become oxidised with tau = 30 micros, concommitant with the reduction of the primary donor of the reaction centre. Subsequently, the alpha-peak of the photooxidised heme broadened and shifted towards longer wavelengths (tau = 70 micros) indicating equilibration of the positive charge over two differing heme species. In the time domain t > 1 ms, rereduction of c-type hemes was seen to be paralleled by a blue shift and further broadening of the alpha-peak. Concommitantly, b-type hemes were observed to first become reduced (within a few milliseconds), then over-oxidised (t > 200 ms) and eventually rereduced to their redox state prior to the flash. The results obtained are discussed with respect to the question of the identity of the immediate electron donor to the photosynthetic reaction centre and with respect to the involvement of a cytochrome bc complex in photo-induced electron transport of green sulphur bacteria.

Chlorobi↗
PubMed1997

Function-directed mutagenesis of the cytochrome b6f complex in Chlamydomonas reinhardtii: involvement of the cd loop of cytochrome b6 in quinol binding to the Q(o) site.

The FUD2 mutant from the green alga Chlamydomonas reinhardtii expresses a cytochrome b6 variant of higher apparent molecular mass [Lemaire et al. (1986) Biochim. Biophys. Acta 851, 239-248]. Here, we show that the mutation corresponds to a 36 base pair duplication in the chloroplast petB gene, which corresponds to a 12 amino acid duplication in the cd loop of cytochrome b6. The resulting protein still binds its heme cofactors and assembles into cytochrome b6f complexes, which accumulate in wild type amounts in exponentially growing cells of FUD2. However, these cytochrome b6f complexes show loosened binding of the Rieske protein and are more prone to degradation in aging cells. Electron transfer through the cytochrome b6f complexes is about 8 times slower in FUD2 than in wild type cells. This is due to a slower oxidation of plastoquinol at the Q(o) site, the folding of which is most likely altered by the duplication. By varying the redox state of the plastoquinone pool in vivo, we show that there is a dramatic decrease in the affinity of the Q(o) site for plastoquinols, which is about 100 times lower in FUD2 than in wild type cells. Our results show that the value of the binding constant of plastoquinol to the Q(o) site (2 x 10(4) M(-1)) derived in [Kramer et al. (1994) Biochim. Biophys. Acta 1184, 251-262] may be extrapolated to in vivo conditions.

Animals↗
PubMed1996

The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.

Amino Acid Sequence↗
PubMed1995

Isolation of a psaF-deficient mutant of Chlamydomonas reinhardtii: efficient interaction of plastocyanin with the photosystem I reaction center is mediated by the PsaF subunit.

The PsaF polypeptide of photosystem I (PSI) is located on the lumen side of the thylakoid membrane and its precise role is not yet fully understood. Here we describe the isolation of a psaF-deficient mutant of the green alga Chlamydomonas reinhardtii generated by co-transforming the nuclear genome of the cw15-arg7A strain with two plasmids: one harboring a mutated version of the psaF gene and the other containing the argininosuccinate lyase gene conferring arginine prototrophy. This psaF mutant still assembles a functional PSI complex and is capable of photoautotrophic growth. However, electron transfer from plastocyanin to P700+, the oxidized reaction center chlorophyll dimer, is dramatically reduced in the mutant, indicating that the PsaF subunit plays an important role in docking plastocyanin to the PSI complex. These results contrast with those obtained previously with a cyanobacterial psaF-, psaJ- double mutant where no phenotype was apparent.

Animals↗
PubMed1995

Conversion of cytochrome f to a soluble form in vivo in Chlamydomonas reinhardtii.

We introduced a stop codon in place of the ATT codon encoding Ile283 (numbered from the Met initiation codon) in the petA gene from Chlamydomonas reinhardtii. The resulting protein was expected to be truncated on its carboxy-terminus end, lacking the last 35 amino acids. This region of the polypeptide sequence encompasses a hydrophobic stretch assumed to anchor the protein in the thylakoid membrane. Once introduced in whole cells of C. reinhardtii by chloroplast transformation, the modified petA gene expressed a truncated apoprotein which was efficiently converted to a truncated holocytochrome f. This protein accumulated in the lumen of the thylakoids in a soluble form. Thus the conversion of preapocytochrome f to holocytochrome f does not require an interaction with the membrane through its C-terminus anchor. We show that the rest of the cytochrome b6f complex failed to accumulate in the transformants, most probably because of a lack of interaction between soluble cytochrome f and the other cytochrome b6f subunits. However, soluble cytochrome f was still able to donate electrons to photosystem I, which is indicative of its ability to maintain interactions with plastocyanin. The control of the rate of synthesis of cytochrome f by the neighboring subunit, suIV (Kuras & Wollman (1994) EMBO J. 13, 1019-1027), was not observed with the truncated cytochrome f. This observation suggests that either the transmembrane anchor of cytochrome f contains a target for the regulation of cytochrome f translation by suIV or there is a transient form of membrane-bound cytochrome f which is highly sensitive to proteolysis at an early post-translational stage.

Amino Acid Sequence↗
PubMed1994

Mechanism of electron transfer in the cytochrome b/f complex of algae: evidence for a semiquinone cycle.

The most widely accepted mechanism of electron and proton transfer within the cytochrome (Cyt) b/f complex derives from the Q-cycle hypothesis originally proposed for the mitochondrial Cyt b/c1 complex by Mitchell [Mitchell, P. (1975) FEBS Lett. 57, 135-137]. In chloroplasts, the Cyt b/f complex catalyzes the oxidation of a plastoquinol at a site, Qo (the plastoquinol binding site), close to the inner aqueous phase and the reduction of a quinone at a site, Qi (the plastoquinone binding site), close to the stromal side of the membrane. In an alternative model, the semiquinone cycle [Wikström, M. & Krab, K. (1986) J. Bioenerg. Biomembr. 18, 181-193], a charged semiquinone formed at site Qo is transferred to site Qi where it is reduced into quinol. Flash-induced kinetics of the redox changes of Cyt b and of the formation of a transmembrane potential have been measured in Chlorella sorokiniana cells incubated in reducing conditions that induce a full reduction of the plastoquinone pool. The experiments were performed in the presence of an uncoupler that collapses the permanent electrochemical proton gradient and thus accelerates the rate of the electrogenic processes. The results show that the electrogenic reaction driven by the Cyt b/f complex precedes the processes of reduction or oxidation of the b-hemes. This electrogenic process is probably due to a transmembrane movement of a charged semiquinone, in agreement with the semiquinone-cycle hypothesis. This mechanism may represent an adaptation to reducing conditions when no oxidized quinone is available at the Qi site.

Journal Article↗
PubMed1991

Restricted diffusion in photosynthetic membranes.

The structural organization of membrane proteins and their linkage by diffusion are topics of much debate. Functional studies in photosynthetic membranes, where rapid equilibration of electron transport between redox centers appears restricted to isolated domains, shed new light on the subject.

Cell Membrane↗
PubMed1990

Electron transfer between primary and secondary donors in Rhodospirillum rubrum: evidence for a dimeric association of reaction centers.

Light-induced oxidation of the primary electron donor P and of the secondary donor cytochrome c2 was studied in whole cells of Rhodospirillum rubrum in the presence of myxothiazole to slow down their reduction. 1. The primary and secondary electron donors are close to thermodynamic equilibrium during continuous illumination when the rate of the electron transfer is light-limited. This implies a long-range thermodynamic equilibration involving the diffusible cytochrome c2. A different behavior is observed with Rhodobacter sphaeroides R26 whole cells, in which the cytochrome c2 remains trapped within a supercomplex including reaction centers and the cytochrome b/c complex [Joliot, P., et al. (1989) Biochim. Biophys. Acta 975, 336-345]. 2. Under weak flash excitation, the reduction kinetics of the photooxidized primary donor are nearly exponential with a half-time in the hundred microseconds time range. 3. Under strong flash excitation, the reduction of the photooxidized primary donor follows a second-order kinetics. About half of the photooxidized primary donor is reduced in a few milliseconds while the remainder stays oxidized for hundreds of milliseconds despite an excess of secondary donors in their reduced form. The flash intensity dependence of the amplitude of the slow phase of P+ reduction is proportional to the square of the fraction of reaction centers that have undergone a charge separation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1980

Dependence of Delayed Luminescence upon Adenosine Triphosphatase Activity in Chlorella.

Delayed luminescence and fluorescence yield after illumination by a short flash were measured in Chlorella pyrenoidosa in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Addition of tri-N-butyl-tin (TNBT), a specific inhibitor of ATPase, drastically increases the life-time of the reduced photosystem II primary acceptor Q(-) and decreases the intensity of delayed luminescence. This indicates a slowing of the charge recombination between the oxidized donor and reduced acceptor of photosystem II centers. No inhibition is observed in isolated chloroplasts when the membrane is permeable to ions, i.e. in the presence of Gramicidin D and KCl.It is suggested that there exists in dark-adapted algae a permanent proton gradient which stimulates the charge recombination process. This proton gradient results from the hydrolysis of a pool of ATP by membrane-bound ATPases and collapses after the addition of TNBT. The long lifetime of this proton gradient (several hours) indicates that the ATP probably comes from the mitochondria.The rate of the back reaction occurring from state S(3) (as defined by Kok, Forbush, and McGloin 1970 Photochem Photobiol 11: 457-475) is more dependent upon the pH gradient than for state S(2).

Journal Article↗