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1.
Experimental evidence for electron transfer, photosensitized by bacteriochlorophyll, from cytochrome c to a pigment complex P-760 (involving bacteriopheophytin-760 and also bacteriochlorophyll-800) in the reaction centers of Chromatium minutissimum has been described. This photoreaction occurs between 77 and 293 degrees K at a redox potential of the medium between -250 and -530 mV. Photoreduction of P-760 is accompanied by development of a wide absorption band at 650 nm and of an EPR signal with g=2.0025+/-0.0005 and linewidth of 12.5+/-0.5 G, which are characteristic of the pigment radical anion. It is suggested that the photoreduction of P-760 occurs under the interaction of reduced cytochrome c with the reaction center state P+-890-P--760 which is induced by light. The existence of short-lived state P+-890-P--760 is indicated by the recombination luminescence with activation energy of 0.12 eV and t 1/2 less than or equal to 6 ns. This luminescence is exicted and emitted by bacteriochlorophyll and disappears when P-760 is reduced. At low redox potentials, the flash-induced absorbance changes related to the formation of the carotenoid triplet state with t 1/2 = 6 mus at 20 degreesC are observed. This state is not formed when P-760 is reduced at 293 and 160 degrees K. It is assumed that this state is formed from the reaction center state P+-890---760, which appears to be a primary product of light reaction in the bacterial reaction centers and which is probably identical with the state PF described in recent works.  相似文献   

2.
The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.  相似文献   

3.
Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studies using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P+) of the primary electron-donor (P) and the photoreduced form (X MINUS) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P+ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P+X minus, and (c) upon transfer of an electron from X minus to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P+ by cytochromes and the oxidation of X minus by Y, the decay kinetics of delayed fluorescence are identical with those of P+X minus, as measured from optical absorbance changes. The main decay route for P+X minus under these conditions has a rate-constant of approximately 10-3-s-minus 1. In contrase, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P+X minus returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s-minus 1, at 295 degrees K and pH 7.8. The decay kinetics of P+X minus and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal mol-mol- minus 1. We conclude that the main decay route involves tunneling of an electron from X minus to P+, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P+X minus results in increases of both the energy and the entropy of the system, by 16.6 kcal-mol- minus 1 and 8.8 cal-mol- minus 1-deg- minus 1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.  相似文献   

4.
An attempt is made to reveal the relation between the induction curves of delayed fluorescence (DF) registered at 0.35-5.5 ms and the prompt chlorophyll fluorescence (PF). A simple formulation was proposed to link the ratio of the transient values of delayed and variable fluorescence with the redox state of the primary electron acceptor of Photosystem II--QA, and the thylakoid membrane energization. The term luminescence potential (UL) was introduced, defined as the sum of the redox potential of QA and the transmembrane proton gradient. It was shown that UL is proportional to the ratio of DF to the variable part of PF. The theoretical model was verified and demonstrated by analysing induction courses of PF and millisecond DF, simultaneously registered from leaves of barley--wild-type and the chlorophyll b-less mutant chlorina f2. A definitive correlation between PF and DF was established. If the luminescence changes are strictly due to UL, the courses of DF and PF are reciprocal and the millisecond DF curve resembles the first derivative of the PFt function.  相似文献   

5.
Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.  相似文献   

6.
Pulse-radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is -(480 +/- 30) mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2 approximately equal to 10(5)S-1) the intermediate state decays to the stable semiquinone form of flavodoxin. The delta G of the conformational change at pH 8 is about 29 kJ mol -1 (0.3 eV). This means that the upper limit for the pK of N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mol -1 (0.45 eV). The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion-controlled with a forward rate constant of (7 +/- 1) X 10(8) M -1S -1 (pH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 +/- 5) mV. A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two-electron process, the midpoint potential of which should be located between -440 mV less than Em (Fld/FldH-) less than -290 mV. The exact value will depend on the delta G of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two-electron donor.  相似文献   

7.
《Inorganica chimica acta》1988,142(2):321-327
The uranyl excited-state lifetimes and luminescence spectra have been examined for a series of bis- triketonate and bis-tetraketonate uranyl— transition metal complexes at low temperatures. The energies of the vibronic components of the uranyl luminescence were found to be dependent on the complexing ligand, but they did not depend significantly on the neighboring transition metal (Cu, Co, Fe, Ni, Zn, Pd). The band shape was sometimes markedly dependent on the metal. Emission quantum yields varied over a 100-fold range. Emission lifetimes varied by less than a factor of three, despite the fact that most of the transition metals are potential quenchers, and despite the existence of a low energy ligand-to-metal charge-transfer excited state in the tetraketonate complexes. The vibronic isolation of the uranyl excited state from other molecular excited states in these complexes is attributed to a large nuclear reorganizational barrier for entry into or escape from the potential energy surface of the electronically excited uranyl moiety. Population of the uranyl excited state results in an increase in the UO bond length, and the UO nuclear motions are not activated by other low energy electronic excited states of the polyketonate complexes.  相似文献   

8.
The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).  相似文献   

9.
Delayed luminescence was measured from samples of a synchronously growing cell culture of the unicellular green alga, Scenedesmus obtusiusculus Chod., every second hour during the 24 h cell cycle under a 15/9 h lighi/dark regime. Both high (air + 2.5% CO2) and low (0.03% CO2) CO2 conditions were used. Under high CO2 conditions, while light excitation induces formation of a late (maximum reached after 10–60 s) transient peak in delayed luminescence in cells sampled after 10–16 h in the cell cycle. During most of the cell cycle low CO2 conditions stimulate a late transient peak formation. Excitation with 700 nm light, but not with 680 nm light, induces a late transient peak in delayed luminescence under high CO2 conditions. The transient peak is more or less pronounced depending on the cell stage. The variations might be due to a changing capacity for light-induced state I/stale II transitions during the cell cycle. It is assumed that the formation of a late transient peak in delayed luminescence is due to ATP hydrolyzation and is thus favoured by a high ATP/NADPH ratio. Hydrolyzation of ATP affects the transthylakoidal ΔpH, which regulates the reverse electron flow to the plastoquinone-pool and QA/QB, thus affecting the decay kinetics of the delayed luminescence.  相似文献   

10.
Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.  相似文献   

11.
Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.  相似文献   

12.
Light is an elusive substrate for the function of photosynthetic light reactions of photosynthesis in the thylakoid membrane. Therefore structural and functional dynamics, which occur in the timescale from seconds to several days, are required both at low and high light conditions. The best characterized short-time regulation mechanism at low light is a rapid state transition, resulting in higher absorption cross section of PSI at the expense of PSII. If the low light conditions continue, activation of the lhcb-genes and synthesis of the light-harvesting proteins will occur to optimize the functions of PSII and PSI. At high light, the transition to state 2 is completely inhibited, but the feedback de-excitation of absorbed energy as heat, known as the energy-dependent quenching (q(E)), is rapidly set up. It requires, at least, the DeltapH-dependent activation of violaxanthin de-epoxidase and involvement of the PsbS protein. Another crucial mechanism for protection against the high light stress is the PSII repair cycle. Furthermore, the water-water cycle, cyclic electron transfer around PSI and chlororespiration are important means induced under high irradiation, functioning mainly to avoid an excess production of reactive oxygen species.  相似文献   

13.
A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state delta pH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by subsaturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the delta pH. This indicates that delta pH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton-translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40-50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation.  相似文献   

14.
The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

15.
B.G. De Grooth  H.J. Van Gorkom 《BBA》1981,635(3):445-456
An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.  相似文献   

16.
In the cytoplasm of activated neutrophils and monocytes of blood, as well as microphages and macrophages of festering wounds, the incubation with nitroblue tetrazolium results, in two types of autoluminescence. The blue luminescence as a result of reduced pyridine nucleotides (RP) characterizes the non-active state of phagocytes. The yellow-green luminescence due to oxygenated flavoproteins (OF) signifies the active state of phagocytes. The luminescence intensity ratio of OF to RP reflects the rate of energy output and is the energy index of phagocyte activation.  相似文献   

17.
Delayed fluorescence from Rhodopseudomonas viridis membrane fragments has been studied using a phosphoroscope employing single, short actinic flashes, under conditions of controlled redox potential and temperature. The emission spectrum shows that delayed fluorescence is emitted by the bulk, antenna bacteriochlorophyll. The energy for delayed fluorescence, however, must be stored in a reaction-center complex including the photooxidized form (P+) of the primary electron-donor (P) and the photoreduced form (X?) of the primary electron-acceptor. This is shown by the following observations: (1) Delayed luminescence is quenched (a) at low redox potentials which allow cytochromes to reduce P+ rapidly after the flash, (b) at higher redox potentials which, by oxidizing P chemically, prevent the photochemical formation of P+X?, and (c) upon transfer of an electron from X? to a secondary acceptor, Y. (2) Under conditions that prevent the reduction of P+ by cytochromes and the oxidation of X? by Y, the decay kinetics of delayed fluorescence are identical with those of P+X?, as measured from optical absorbance changes.The main decay route for P+X? under these conditions has a rate-constant of approximately 103 s?1. In contrast, a comparison of the intensities of delayed and prompt fluorescence indicates that the process in which P+X? returns energy to the bulk bacteriochlorophyll has a rate-constant of 3.7 s?1, at 295 °K and pH 7.8. The decay kinetics of P+X? and delayed fluorescence change little with temperature, whereas the intensity of delayed fluorescence increases with increasing temperature, having an activation energy of 12.5 kcal · mol?1. We conclude that the main decay route involves tunneling of an electron from X? to P+, without the promotion of P to an excited state. Delayed fluorescence requires such a promotion, followed by transfer of energy to the bulk bacteriochlorophyll, and this combination of events is rare. The activation energy, taken with potentiometric data, indicates that the photochemical conversion of PX to P+X? results in increases of both the energy and the entropy of the system, by 16.6 kcal · mol?1 and 8.8 cal · mol?1 · deg?1. The intensity of delayed fluorescence depends strongly on the pH; the origin of this effect remains unclear.  相似文献   

18.
The hypothesis that Na+ and K+ gradients have an energy storing function [V. P. Skulachev (1978) FEBS Lett. 87, 171-176] has been tested in experiments with Escherichia coli, the marine bacterium Vibrio harveyi, an extremely halophilic Halobacterium halobium and a fresh-water cyanobacterium Phormidium uncinatum from Lake Baikal living at an extremely low salt concentration. The capability of these microorganisms to maintain delta microH was compared using motility as a delta microH-supported function. It was found that in all cases the gradient of monovalent cations is competent to prolong the period of active motility after other energy sources are exhausted. Maximal prolongation was found in H. halobium, which in a Na+ medium was still motile when light was switched off for 9 h under anaerobic conditions. In V. harveyi the motility was maintained for 1 h, in E. coli for about 10 min and in Ph. uncinatum for about 2 min. Thus the delta microH buffer capacity of the monovalent cation gradient is proportional to the content of these cations in the habitat. It was also found that in Ph. uncinatum only delta pK is effective, whereas in E. coli and V. harveyi both delta pK and delta pNa are. In E. coli when the K+ release is completed and the cells become motionless, motility can be temporarily restored by adding NaCl which initiates an H+ efflux. Under conditions of exhaustion of energy sources, the Na+ and K+ gradient was shown to stabilize potential in H. halobium cells, measured with a tetraphenylphosphonium probe. In H. halobium and E. coli, the anaerobic ATP level was found to stabilize when the Na+ and K+ gradients were present. Addition of N,N'-dicyclohexylcarbodiimide destabilized this level, which indicated that Na+ and K+ gradients could support de novo ATP synthesis. It is concluded that the data obtained are in agreement with the concept of the energy storing by the Na+ and K+ gradients. Other functions of these gradients and the mechanisms of their formation are discussed.  相似文献   

19.
Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.  相似文献   

20.
The 90-residue N-terminal Phox and Bem1p (PB1) domain of NBR1 forms an α/β ubiquitin-like fold. Kinetic analysis using stopped-flow fluorescence reveals two-state kinetics; however, nonlinear effects in the denaturant dependence of the unfolding data demonstrate changes in the position of the rate-limiting barrier along the folding coordinate as the folding conditions change. The kinetics of wt-PB1 and several mutants show that this curvature is consistent with a single-pathway mechanism involving sequential transition states (TS1 and TS2) separated by a transiently populated high-energy intermediate, rather than movement of the transition state on a broad energy plateau. We show that the two transition states within the sequential model represent structurally and thermodynamically distinct species. TS1 is a collapsed state (αTS1 = 0.71) with a large enthalpic barrier to formation that is rate-limiting under conditions that strongly favour folding. TS2 is highly native-like (αTS2 = 0.93) and represents a late entropic barrier to formation of the native state. In support of the sequential transition state mechanism, we show that the G62A helix 2 substitution stabilises TS1 and the intermediate to such an extent that the latter becomes significantly populated, leading to the observation of a fast kinetic phase representing the initial U → I transition, with TS2 (αTS2 = 0.87) becoming rate-limiting. The folding rate is not retarded by populating an intermediate, which would be expected for a misfold state, but is accelerated, suggesting that the I state is productive and on-pathway. The results show that the apparent two-state folding of the wt-PB1 domain occurs along a well-defined pathway involving structurally and thermodynamically distinct sequential transition states and an obligatory metastable intermediate that represents a productive local minimum in the energy landscape that increases the efficiency of barrier crossing through favourable effects on the entropy of activation.  相似文献   

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