Biomedical subjects
Publications and source records attributed to P Joliot.
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.
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)
The C550 absorption change and the fluorescence yield were studied at room temperature in chloroplasts in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and under conditions in which contributions of P-700 and of the electrochromic effect were neglible. 1. The C550 difference spectrum is a typical band shift with an isobestic point close to 550 nm. 2. The maximum amplitude of C550 absorption change is reached upon the first flash of a series of saturating flashes, unlike the maximum fluorescence yield which is attained after several flashes. 3. The comparison of the induction curves of the C550 change and the fluorescence yield in weak light shows that the fluorescence yield is controlled by two quenchers: one of them (Q1), the redox state of which C550 is a probe, is responsible for the major part of the quenching; the other one (Q2), which is less concentrated and less efficient becomes predominant at the end of the fluorescence induction. 4. Quencher Q2 back-reacts faster than quencher Q1. 5. Two alternative models are discussed in which Q1 and Q2 belong either to the same Photosystem II center or to two different photocenters.
Flash-induced transients in light scattering were shown to occur with Chlorella cells. The kinetic and spectral patterns of the scattering transients and their relation to the absorption changes studied in the 10 microseconds--5 s time range, between 450 and 540 nm. 1. The kinetics of the fast changes (less than 500 ms) in scattering and absorbance were identical. From about 500 ms divergence of the two signals was observed. 2. The transient spectrum characterizing the fast scattering changes exhibited a large double band between 480 and 500 nm. Transients corresponding to the slower changes resembled the steady scattering spectrum (Latimer, P. and Rabinowitch, E. (1959) Arch. Biochem. Biophys. 84, 428--441) with a maximum at about 515 nm. 3. From theoretical considerations it is suggested that fast transients in scattering and absorbance are physically interrelated, and as has been shown for absorption changes (Witt, H.T. (1971) Q. Rev. Biophys. 4, 365--477) fast scattering transients can also be interpreted as an electrochromic phenomenon. Slower changes are accounted for by alterations in the microenvironment and conformation of the particles responsible for scattering.
The rise kinetics of the absorption changes induced at 515 nm and 480 nm by a flash were studied using two types of xenon flashes of different durations. The 'slow' rise of the absorption change (t 1/2 = 15--20 microseconds) observed by Cox and Delosme (1978 C.R. Acad. Sci. (Paris) Sér. D 282, 775--778) and Joliot was found to be due to double hits occurring in the reaction centers of System I during the flash. The turnover kinetics of the reaction centers of System I after a short flash were studied by a double flash method. They are in agreement with a second order reaction between P+-700 and its electron donor.
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1. The amplitudes of the fast (0-20 microseconds) and slow (20 microseconds-2 ms) fluorescence rise induced by a 2 microseconds flash have been measured as a function of the energy of the flash in chloroplasts inhibited by 3(3,4-dichlorophenyl)-1, 1-dimethylurea. The saturation curve for the slow rise shows a characteristic lag which is not observed for the fast fluorescence rise. This lag indicates that Photosystem II centers undergo a double hit process which implies that (a), each photocenter includes two acceptors Q1 and Q2; (B), after the first hit, oxidized chlorophyll Chl+ is reduced by a secondary acceptor Y in a time shor compared to the duration of the flash; (c), after the second hit, Chl+ is reduced by another secondary donor, D. 2. According to Den Haan et al. (1974) Biochim. Biophys. Acta 368, 409-421), hydroxylamine destroys the secondary donor responsible for the fast reduction of Chl+. In the presence of 3 mM hydroxylamine, only the secondary donor D is functional and a flash induses mainly a single hit process. 3. The saturation curves for the fast and the slow rises have been studied in the presence of 3(3,4-dichlorophenyl)-1, 1-dimethylurea for a second actinic flash given 2.5 s after a first saturating one. The large decrease in the half-saturating energy indicates the existence of efficient energy transfer occuring between potosynthetic units. 4. Two alternate hypotheses are discussed (a) in which D is an auxiliary donor and (b) in which D is included in the main electron transfer chain.
Using Chlorella, three types of absorption changes at 515 nm have been studied in the 4-100 mus time range following a flash. (1) The absorption change observed when both photoreactions are blocked, probably due to the formation of the triplet state of a carotenoid, is show to depend on Photosystem II excitation only. (2) The absorption increase induced by photoreaction I is biphasic; first phase, complete in less than 4 mus, followed by a slower phase with a half-rise time of 15-20 mus. (3) On the other hand, photoreaction II induces only a fast absorption increase (lessthan 4 mus). The time course of the biphasic 515 nm absorption increase induced by photoreaction I is similar to the biphasic absorption decrease previously observed at 480 nm by Cox and Delosme (1976, C.R. Acad. Sci. Paris 282D, 775-778). No significant absorption change is observed at 490 nm. These results suggest that the transmembrane electric field induced by photoreaction I rises to its maximum value in at least two phases within 100 mus following flash excitation.
The intermediate phase of fluorescence relaxation (lms-ls) (Joliot, P., Joliot, A., Bouges, B, and Barbieri, G. (1971) Photochem. Photobiol. 14, 287-305), following a single saturating flash, is shown to be controlled by a slow phase of the reoxidation of Q- by a secondary acceptor and, in vivo, by the transmembrane electric field. The kinetics of reoxidation of Q- are slowed by lowering the pH. This slowing effect is interpreted in terms of the reversible formation at low pH of QH which is not oxidizable by the secondary acceptor. The electric field transforms Photosystem II centers into a non-quenching photochemically inactive state that cannot be attributed to an accumulation of Q-. Centers are unequally sensitive to the field. A critical field strength can be defined for each center above which that center is blocked and below which the center is photochemically active. The transformation from the active to inactive state occurs over a narrow range of field strength. Sensitive centers are blocked by the field in less than 1 ms and become active again in less than 10 ms as the field strength falls. Two hypotheses are proposed for the mechanism of blockage of centers by the field: (1) a field induced conformational change in the centers, (2) the formation or suppression of a dipole critical to the function of a center. The activity of the ATP synthetase, determining the rate of relaxation of the field, was controlled by a light-dark treatment or by a chemical method using p-benzoquinone.
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The concept of photosynthetic unit (PSU) is reviewed in the light of the authors' results in the fields of fluorescence and luminescence (delayed light). Models of PSU are mainly distinguished by the amount of exciton exchange which is allowed between units. The "separate" model, with its "first-order" character, is not consistent with fluorescence kinetic data. The sigmoidal rise of fluorescence under actinic light is best explained by "nonseparate" models; however, most of these models assume a delocalization of excitons or centers. The "connected" model introduced here is not subject to this criticism. It discloses a new effect (the "îlot" effect): a nonrandom grouping of fluorescent units the consequences of which are discussed. It is noted that a "two-quantum" model for the photochemical reaction gives results very similar to those of the connected model. A relation between luminescence intensity and fluorescence yield is seen as a necessary consequence of the PSU concept. Its meaning is different in separate and nonseparate models. This relation is discussed in connection with the true system II fluorescence emission.
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