Hydroxyectoine protects Mn-depleted photosystem II against photoinhibition acting as a source of electrons.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K4[Fe(CN)6], DPC, TMPD, Fe2+, and Mn2+) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment-protein complexes.

Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR.

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Labile assembly of a tardigrade protein induces biostasis.

Tardigrades are microscopic animals that survive desiccation by inducing biostasis. To survive drying tardigrades rely on intrinsically disordered CAHS proteins, which also function to prevent perturbations induced by drying in vitro and in heterologous systems. CAHS proteins have been shown to form gels both in vitro and in vivo, which has been speculated to be linked to their protective capacity. However, the sequence features and mechanisms underlying gel formation and the necessity of gelation for protection have not been demonstrated. Here we report a mechanism of fibrillization and gelation for CAHS D similar to that of intermediate filament assembly. We show that in vitro, gelation restricts molecular motion, immobilizing and protecting labile material from the harmful effects of drying. In vivo, we observe that CAHS D forms fibrillar networks during osmotic stress. Fibrillar networking of CAHS D improves survival of osmotically shocked cells. We observe two emergent properties associated with fibrillization; (i) prevention of cell volume change and (ii) reduction of metabolic activity during osmotic shock. We find that there is no significant correlation between maintenance of cell volume and survival, while there is a significant correlation between reduced metabolism and survival. Importantly, CAHS D's fibrillar network formation is reversible and metabolic rates return to control levels after CAHS fibers are resolved. This work provides insights into how tardigrades induce reversible biostasis through the self-assembly of labile CAHS gels.

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift.

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (-11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (-8.2 kcal/mol) and the yield in the first flash is already one half of the maximum, which is reached after 2-3 turnovers. 2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing delta psi is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold. 3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (td) between flashes, with an optimum for td = 160-320 ms. 4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (-10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10(-8) M valinomycin. 5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis. 6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash. 7. The conclusions drawn are independent from the assumption that a delta psi between bulk phases is evaluable from the carotenoid signal.

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

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

Photosynthetic control and estimation of the optimal ATP: electron stoichiometry during flash activation of chromatophores from Rhodopseudomonas capsulata.

(1) When chromatophores from Rhodopseudomonas capsulata Ala pho+ are exposed to a train of high-frequency, saturating flashes the kinetics of the reaction centre bacteriochlorophyll absorption change enter a pseudo steady-state in which the extent of oxidation during the flashes is equal to the extent of reduction in between the flashes. The level of the pseudo steady-state is lowered by the presence of a phosphate acceptor system, raised by further addition of oligomycin, lowered by a combination of nigericin and valinomycin and raised by antimycin A. (2) In the pseudo steady-state, the extent of reaction centre bacteriochlorophyll oxidation taking place during the flash may be estimated by subtraction from the total concentration of reaction centre bacteriochlorophyll. This value is equated with the amount of electrons transported through the photosynthetic chain. Comparison with the measured ATP yield per flash in the pseudo steady-state permits calculation of the ATP: two electron ratio. The value of the ratio is 1.1 for flash frequencies between 3 and 12.5 Hz and declines at lower and higher frequencies. The ATP: two electron ratio is approximately halved in the presence of antimycin A. (3) An alternative estimate of the ATP: two electron ratio, based on the assumption that high-frequency flashes approximate to the condition of continuous illumination, was approx. 0.8.

A minimal hypothesis for membrane-linked free-energy transduction. The role of independent, small coupling units.

Experimental data are reviewed that are not in keeping with the scheme of 'delocalized' protonic coupling in membrane-linked free-energy transduction. It turns out that there are three main types of anomalies: (i) rates of electron transfer and of ATP synthesis do not solely depend on their own driving force and on delta mu H, (ii) the ('static head') ratio of delta Gp to delta mu H varies with delta mu H and (iii) inhibition of either some of the electron-transfer chains or some of the H+-ATPases, does not cause an overcapacity in the other, non-inhibited proton pumps. None of the earlier free-energy coupling schemes, alternative to delocalized protonic coupling, can account for these three anomalies. We propose to add a fifth postulate, namely that of the coupling unit, to the four existing postulates of 'delocalized protonic coupling' and show that, with this postulate, protonic coupling can again account for most experimental observations. We also discuss: (i) how experimental data that might seem to be at odds with the 'coupling unit' hypothesis can be accounted for and (ii) the problem of the spatial arrangement of the electrical field in the different free-energy coupling schemes.

The high potential iron-sulfur protein (HiPIP) from Rhodoferax fermentans is competent in photosynthetic electron transfer.

The functional role of the High Potential Iron-sulfur Protein (HiPIP) from the photosynthetic bacterium Rhodoferax fermentans was investigated. We demonstrated that the HiPIP increased the rate of light-induced oxygen reduction mediated by the photosynthetic reaction center (RC); this stimulation reached half-saturation at [HiPIP]/[RC] ca. 15. The capability of the HiPIP in delivering electrons to the reaction center of Rhodoferax fermentans was demonstrated through kinetic spectrophotometry of cytochrome c-556 oxidation in the presence or in the absence of HiPIP. It is concluded that the HiPIP is competent in the photosynthetic electron transfer chain of Rhodoferax fermentans.

Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria. A method of correction for the electrochromic effects.

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self-consistency of the method. The corrected traces for reaction center and of cytochrome b-566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will be possible to study the interrelation between proton gradient and individual electron reactions under flash or steady-state illumination.

Interaction between cytochrome c and the photosynthetic reaction center of purple bacteria: behaviour at low temperature.

In purple photosynthetic bacteria the electron donor to the special pair, after its oxidation by a light-induced reaction, is a c-type cytochrome: either a soluble monoheme cytochrome which forms a transitory complex with the reaction center, or a tetraheme cytochrome which remains permanently bound to the reaction center. The effects of low temperatures on electron transfer in the complex are presented and discussed. They provide estimates for the reorganization energy. The most prominent effect of low temperature is that a dominant fast phase of electron transfer becomes impossible at a temperature of around 250 K (monoheme cytochrome) or located between 250 K and 80 K according to the redox state (tetraheme cytochrome). This inhibition is attributed to a freezing-like transition of pools of water molecules which blocks structural changes of the protein which are normally associated with the cytochrome oxidation.

Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX-.

Low-temperature (1.4 K), single-molecule fluorescence-excitation spectra have been recorded for individual reaction center-light-harvesting 1 complexes from Rhodopseudomonas palustris and the PufX(-) strain of Rhodobacter sphaeroides. More than 80% of the complexes from Rb. sphaeroides show only broad absorption bands, whereas nearly all of the complexes from Rps. palustris also have a narrow line at the low-energy end of their spectrum. We describe how the presence of this narrow feature indicates the presence of a gap in the electronic structure of the light-harvesting 1 complex from Rps. palustris, which provides strong support for the physical gap that was previously modeled in its x-ray crystal structure.

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.

Electron transfer from cytochrome c2 to the primary donor of Rhodobacter sphaeroides reaction centers. A temperature dependence study.

Kinetics of flash-induced electron transfer from the soluble cytochrome c2 to the primary donor (P) of the reaction center purified from the purple bacterium Rhodobacter sphaeroides R-26 were investigated by time-resolved absorption spectroscopy. Re-reduction of P+ induced by a laser pulse was measured at 1283 nm both in isolated reaction centers and in reconstituted proteoliposomes reproducing the lipid composition of the native membrane. The effects of temperature (230-300 K) and of the cytochrome c2/reaction center stoichiometry were examined. At room temperature, over a wide range of cytochrome c2 to reaction center molar ratios, the biphasic kinetics of cytochrome c2 oxidation in the microsecond-to-millisecond time scale could be accurately described by a minimum reaction scheme which includes a second-order collisional process (k = 1.4 x 10(9) M-1 s-1 and k = 2.4 x 10(9) M-1 s-1 in isolated and reconstituted reaction centers, respectively) and a first-order intracomplex electron donation (t1/2 = 590 +/- 110 ns in isolated reaction centers; t1/2 = 930 +/- 140 ns in proteoliposomes). At cytochrome c2 to reaction center molar ratios exceeding 5, the monomolecular process almost completely accounts for P+ re-reduction. At lower stoichiometries, the relative contribution of the two parallel reaction pathways is modulated by a single binding equilibrium between cytochrome c2 and reaction centers, yielding a binding constant of 3.5 x 10(5) M-1 in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)

A critical evaluation of the hydropathy profile of membrane proteins.

New membrane-preference scales are introduced for categories of membrane proteins with different functions. A statistical analysis is carried out with several scales to verify the relative accuracy in the prediction of the transmembrane segments of polytopic membrane proteins. The correlation between some of the scales most used and those calculated here provides criteria for selecting the most appropriate methods for a given type of protein. The parameters used in the evaluation of the hydropathy profiles have been carefully ascertained in order to develop a reliable methodology for hydropathy analysis. Finally, an integrated hydropathy analysis using different methods has been applied to several sequences of related proteins. The above analysis indicates that (a) microsomal cytochrome P450 contains only one hydrophobic region at the N-terminus that is consistently predicted to transverse the membrane: (b) only four of the six or seven putative transmembrane helices of cytochrome oxidase subunit III are predicted and correspond to helices I, III, V and VI of the previous nomenclature; (c) the product of the mitochondrial ATPase-6 gene (or the chloroplast ATPase-IV gene) of F0-F1-ATPase shows that helix IV is not consistently predicted to traverse the membrane, suggesting a four-helix model for this family of proteins.

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.

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center.

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. II. Direct electron transfer between the reaction center and the bc1 complex and role of cytochrome c2.

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.

Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus.

1. The kinetics of decay in the dark of the transmembrane pH difference (delta pH) induced by light in nonphosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9-aminoacridine, in the presence of 50 mM KCl and 2 microM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-228]. The kinetic competence of the probe response was tested by accelerating the delta pH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different delta pH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 micron.s-1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of delta pH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K(+)-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of PufX protein in photosynthetic growth of Rhodobacter sphaeroides. 1. PufX is required for efficient light-driven electron transfer and photophosphorylation under anaerobic conditions.

The pufX gene is essential for photoheterotrophic growth of the purple bacterium Rhodobacter sphaeroides. In order to analyze the molecular function of the PufX membrane protein, we constructed a chromosomal pufX deletion mutant and phenotypically compared it to a pufX+ control strain and to two suppressor mutants which are able to grow photosynthetically in the absence of pufX. Using this genetic background, we confirmed that PufX is required for photoheterotrophic growth under anaerobic conditions, although all components of the photosynthetic apparatus were present in similar amounts in all strains investigated. We show that the deletion of PufX is not lethal for illuminated pufX- cells, suggesting that PufX is required for photosynthetic cell division. Since chromatophores isolated from the pufX- mutant were found to be unsealed vesicles, the role of PufX in photosynthetic energy transduction was studied in vivo. We show that PufX is essential for light-induced ATP synthesis (photophosphorylation) in anaerobically incubated cells. Measurements of absorption changes induced by a single turnover flash demonstrated that PufX is not required for electron flow through the reaction center and the cytochrome bc1 complex under anaerobic conditions. During prolonged illumination, however, PufX is essential for the generation of a sufficiently large membrane potential to allow photosynthetic growth. These in vivo results demonstrate that under anaerobic conditions PufX plays an essential role in facilitating effective interaction of the components of the photosynthetic apparatus.