Extent Of Proton Uptake Research Articles

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon QB photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 → Asn replacement on QB−/QB FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 → Asp, Arg M233 → Cys, or Arg H177 → His suppressor mutation. The proton uptake by Glu L212 upon QB− formation, as reflected by the positive band at 1728 cm−1, is increased in the Asn M44 → Asp and Arg H177 → His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 → Asn mutant RCs. Only the Arg M233 → Cys suppressor mutation affected the 1728 cm−1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 → Asn lesion.

Effect of uncouplers on the bioenergetic properties of a carbonyl cyanide m-chlorophenylhydrazone-resistant mutant Escherichia Coli UV6

The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and tri- n-butyltin chloride (Bu 3SnCl) on proline transport, proton uptake and the transmembrane pH gradient in intact cells have been compared in a CCCP-resistant mutant strain Escherichia coli UV6, and its parent strain, AN180. CCCP and Bu 3SnCl inhibited proline uptake in AN180 but the pH gradient was affected only by CCCP. Neither uncoupler affected the pH gradient in UV6 although inhibition of proline uptake occurred at high concentrations. CCCP caused efflux of accumulated proline in both strains but Bu 3SnCl was ineffective. Bu 3SnCl did not prevent the efflux of proline induced by CCCP, indicating that Bu 3SnCl had not inactivated the transport carrier. In contrast with the parent strain, CCCP failed to reverse the oxidation-dependent inhibition of the phosphotransferase system in UV6 even at concentrations causing inhibition of proline uptake. The phosphorylation potential of UV6 with succinate as substrate was lower than in AN180. This was associated with a 10-fold higher concentration of phosphate in succinate-grown UV6 than in AN180. These results suggest that CCCP and Bu 3SnCl have different sites of action on the membrane energization system of intact cells of E. coli. A possible explanation of the differences between AN180 and UV6 is that the energization system is altered in the CCCP-resistant mutant. Both uncouplers stimulated the uptake of protons by intact cells to the same extent in UV6 as in AN180. In UV6, and in AN180 with Bu 3SnCl, this was not accompanied by effects on the transmembrane pH gradient. The extent of proton uptake appeared to be related to the level of the highly anionic membrane-associated oligosaccharides in the periplasmic space. It is proposed the outer membrane acts as a partial barrier to protons and that the uncouplers can equilibrate protons between the extracellular medium and the periplasmic space in intact cells.

Proton movement in reconstituted purple membrane of Halobacteria: Effects of pH and ionic composition of the medium

Bacteriorhodopsin has been incorporated into sonicated phospholipid vesicles to form the functionally active proton pump. The kinetics of the light-induced proton uptake and of the subsequent release of protons in the dark have been analyzed according to a scheme which assumes the existence in bacteriorhodopsin of a light-dependent pathway responsible for an inhibition of the proton-pumping activity. The growth stage of proton movement obeys the empirical equation ln(1 − Δ Δ s ) = −k L t , where Δ and Δ s are the extent of proton uptake at time t of illumination and at the steady state, respectively. The release of protons in the dark stage follows the decay equation ln( Δ Δ s ) = −k D t , where k D is a light-independent rate constant. With these definitions, the rate constant for the light-dependent proton-pumping inhibition is ( k L − k D) = k I. The initial proton-pumping rate, R 0, is obtained either directly from the “on rate” or from the steady-state equation R 0 = k LΔ s. We find that, in vesicles made from egg yolk phosphatidylcholine, changes in pH in the range 5–7 result in changes in both k I and R 0, but have no significant effect on k D. The two empirical kinetic parameters, k I and R 0, do not change to the same extent with pH, indicating a pH dependence of the coupling between them. When negatively charged phospholipids, e.g., phosphatidic acid, are incorporated into the phosphatidylcholine vesicles, the inhibition k I and the initial pumping rate, R 0, are sensitive to pH changes. However, there are differences in the pH effects in the presence and absence of acidic phospholipids, and the coupling between pumping and its inhibition is no longer pH dependent. Thus, the presence in the bilayer of phospholipids with negatively charged head groups seems to stabilize the mechanism of coupling between R 0 and k I. Vesicles made from soybean phospholipids have been employed to study the effect of external ionic composition on the kinetics of the lightinduced proton movement. The K + of the external medium is replaced by other monovalent cations, at constant ionic strength and a fixed composition of the internal medium. Replacement of K + by Na + has no effect on the rate constants k D and k D but causes a gradual increase in the initial proton-pumping rate, R 0. Replacement of K + by Cs + causes a marked decrease in the inhibition of the proton pumping measured by k I, without any concomitant effect on the other kinetic parameters. When most of the K + has been replaced by Cs +, pronounced effects are observed in all the parameters. Replacement of K + by Li + has no significant effect on any of the kinetic parameters, except when the K + Li + ratio is very low, in which case pronounced effects are again observed in all the kinetic parameters. These results and previous work from this laboratory, including the modification of the light-dependent proton-pumping inhibition by the charge of the bilayer, support a picture in which there is an indirect link between the primary proton-pumping activity and its associated inhibitory process in bacteriorhodopsin.

Effect of chemical modification of amino groups by fluorescamine on partial reactions of photosynthesis

4-Phenylspiro[furan-2(3H),1-phtalan]3,3′-dione (fluorescamine) was used to covalently modify amino groups of thylakoids. Subsequently its effect on parameters of energy transfer and phosphorylating activity was assessed. While electron transport, the extent of proton uptake, 515 nm change and 9-aminoacridine quench were relatively resistant to such treatment, the functions connected to coupling factor 1, namely ATP formation by acid/base transition, ATPase activity and photophosphorylation were affected much earlier. Photophosphorylation appears to be the most sensitive. The data are interpreted as indicating an involvement of free amino groups in energy transfer.

Effect of temperature on the function of a proton pump.

Vesicles with proton translocating activity were reconstituted with bacterial rhodopsin lipoprotein and synthetic phospholipids. Reconstitution with dimyristoyl phosphatidylcholine yielded vesicles which catalyzed light-driven proton uptake which was sensitive to nigericin, gramicidin or an uncoupler when tested at temperatures above 20°C. At temperatures below 5°C, the extent of proton uptake was actually greater than at 20°C, but there was little effect of either nigericin or uncouplers even when these compounds had been added at 20°C. Gramicidin inhibited at all assay temperatures provided it was added at 20°C. With dipalmitoyl phosphatidylcholine similar results were obtained except that nigericin lost its effectiveness at higher temperatures. On illumination tetraphenylboron was taken up by the reconstituted vesicles. We propose that an electrogenic proton translocation takes place by a channel mechanism.

The relation of millisecond delayed light emission to light-induced ion accumulations in chloroplasts

Delayed fluorescence (delayed light emission) from chloroplasts is increased by ATP, ADP and, to a lesser extent, by ITP. However, neither phosphorylation nor ATP utilization seems to play any part in the phenomenon since the energy transfer inhibitor deoxyphlorizin, which is also an ATPase inhibitor, has no effect on the enhancement of delayed fluorescence. The enhancement of delayed fluorescence by these nucleotides is accompanied by an increase in the extent of proton uptake and n decrease in the nonphosphorylating (basal) electron transport. Uncouplers and ionophores such as imidazole, glycineamide, morpholine, methyl-amine, cyclohexylamine, atebrin, and gramicidin nearly abolish delayed fluorescence. However, ammonium salts are exceptional; they considerably enhance the emission although they also abolish phosphorylation and proton gradient formation. This enhancement of delayed fluorescence occurs only near or above pH 8 and seems to be specific for ammonia when relatively intact lamellae are employed. When particles prepared therefrom with digitonin are used, methylamine also enhances the delayed fluorescence. The enhancement by ammonium salts is correlated with the uptake of ammonium ions. Valinomycin, which is known to increase the permeability of membranes to ammonium ions, abolishes delayed fluorescence in the presence of ammonium salts. It is suggested that (a) ammonia uncoupling abolishes the pH component of the light-induced transmembrane electrochemical potential gradient, but that (b) at higher pH's the electrical component of the gradient (the membrane potential) is not abolished and may even increase while (c) this increased membrane potential is responsible for enhancement of the delayed fluorescence. Gradients which contribute to delayed fluorescence are not necessarily capable of supporting phosphorylation. The requirements for phosphorylation seem more stringent than the requirements for delayed fluorescence and it may be that phosphorylation, unlike the delayed light emission, has an obligatory requirement for a pH gradient.

The Distribution of Photosynthetic Reactions in the Chloroplast Lamellar System

Chloroplast fragmentation, ATPase, Proton pump, Cyclic Phosphorylation, NH4Cl-uncoupling Cyclic phosphorylation and latent ATPase follow the distribution of photosystem I in the chloroplast membrane. Both activities are found higher in stroma lamellae than in grana. The extent of proton uptake is found to be higher in the grana. Since this uptake depends on internal volume and buffer capacity besides proton pump activity, the distribution of the proton pump proper remains to be elucidated. Any fragmentation of the large inner compartment of the chlorolast lamellar system into smaller vesicles results in decreased sensitivity of cyclic phosphorylation to uncoupling by ammonium chloride. Consequently both, isolated grana and stroma lamellae, show decreased uncoupling by ammonium chloride. The effect can be explained by the action of a membrane potential in photophosphorylation which builds up during illumination and might be more stable in a system with the larger number of individual compartments just for statistical reasons. No assumption on changes in specific ion permeabilities during fragmentation of chloroplasts are needed.