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PubMed2000

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant.

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.

Arginine↗
PubMed2004

Degenerin sites mediate proton activation of deltabetagamma-epithelial sodium channel.

The delta-subunit of epithelial Na(+) channels (ENaC) is predominately expressed in brain, heart, and pancreas. The amiloride sensitivity, Na(+) conductance, and critical domains for gating are characterized as a cross between proton-activated Na(+) channels and alpha-ENaC. The hypothesis that external protons may activate human delta-ENaC was addressed by expressing deltabetagamma-hENaC in Xenopus oocytes and evaluating proton-activated current with the two-electrode voltage clamp technique. Our results showed that protons transiently evoked a Na(+) current with an EC(50) of pH 6 overlapped on the basal current of deltabetagamma-hENaC. Proton-activated current was not observed in uninjected oocytes. Studies on gating kinetics revealed that activation, desensitization, and recovery times of proton-activated Na(+) current were 3.8 +/- 0.5 s, 253 +/- 9.5 s, and 10 +/- 3.6 s, respectively (n = 4-12). Alkali metal cation selectivity of the proton-activated current was identical to that of the basal current of deltabetagamma-hENaC. The metabolic acids, lactate, pyruvate, and formate, modified the proton-activated current, as did hypo-osmotic stress. EDTA, hypo-osmolarity, and lactate enhanced proton activation synergistically. Our results suggest that delta-hENaC subunit is essential for proton-activated current and gamma-subunit may potentially regulate the response of delta-hENaC to protons. We have concluded that deltabetagamma-hENaC is a proton-activated cation channel whose closing gate can be regulated by a proton-induced conformational change. Proton-sensitivity of deltabetagamma-hENaC may be an important mechanism for integrating external ischemic signals in inflamed and hypoxic tissues.

Acid Sensing Ion Channels↗
PubMed1991

Tracking of proton flow during transition from anaerobiosis to steady state. 1. Response of matrix pH indicators.

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non-respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of the KCl concentration and is inhibited by Pi and ammonium ions. 2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EGTA-untreated mitochondria, BCECF records an extraction of protons from matrix bulk water of about 2-3 nmol H+/mg, reduced to 1-2 nmol H+/mg in EGTA-treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6-7 nmol H+/mg, it appears that 75-90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red. 3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation. 4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect the distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed.

2,4-Dinitrophenol↗
PubMed1992

THE CHLOROPLAST H+-ATPase: PARTIAL REACTIONS OF THE PROTON.

This article reviews proton intake, charge transfer and proton release by F-ATPases, based in part on flash spectrophotometric studies on the chloroplast ATPase in thylakoid membranes, CF1Fo. The synthesis-coupled translocation of charges by CF1Fo (maximum rate <1500 s-1) and the dissipative flow through its exposed channel portion, CFo (rate >10 000 s-1), are extremely proton-specific (selectivity H+:K+>10(7):1). The proton-specific filter is located in CFo. Proton flow through exposed CFo can be throttled by adding subunit (&dgr;) or subunit &bgr; of CF1. These subunits thus may provide energy-transducing contacts between CF1 and CFo. Recently, we characterized two conditions where, in contrast to the above situation, proton intake by CF1Fo was decoupled from proton transfer across the main dielectric barrier: (a) CF1Fo structurally distorted by low ionic strength transiently trapped protons in a highly cooperative manner, but remained proton tight. This result has been interpreted in terms of Mitchell's proton well. (b) In the absence of nucleotides there is a proton slip. Addition of nucleotides (100 nmol l-1 ADP) abolished proton conduction but not proton intake by CF1Fo. These experiments functionally tag proton binding groups on CF1Fo that are located before the main dielectric barrier.

Journal Article↗
PubMed2006

Proton in the well and through the desolvation barrier.

The concept of the membrane proton well was suggested by Peter Mitchell to account for the energetic equivalence of the chemical (DeltapH) and electrical (Deltapsi) components of the proton-motive force. The proton well was defined as a proton-conducting crevice passing down into the membrane dielectric and able to accumulate protons in response to the generation either of Deltapsi or of DeltapH. In this review, the concept of proton well is contrasted to the desolvation penalty of > 500 meV for transferring protons into the membrane core. The magnitude of the desolvation penalty argues against deep proton wells in the energy-transducing enzymes. The shallow DeltapH- and Deltapsi-sensitive proton traps, mechanistically linked to the functional groups in the membrane interior, seem more realistic. In such constructs, the draw of a trapped proton into the membrane core can happen at the expense of some exergonic reaction, e.g., release of another proton from the membrane into the aqueous phase. It is argued that the proton transfer in the ATP synthase and the cytochrome bc complex could proceed in this way.

Biological Transport↗
PubMed2006

Protons @ interfaces: implications for biological energy conversion.

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from "pulsed" experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the "sources" and "sinks", the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.

Biological Transport↗
PubMed2004

Towards understanding the tandem mass spectra of protonated oligopeptides. 1: mechanism of amide bond cleavage.

The mechanism of the cleavage of protonated amide bonds of oligopeptides is discussed in detail exploring the major energetic, kinetic, and entropy factors that determine the accessibility of the b(x)-y(z) (Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2002, 16, 375) and "diketopiperazine" (Cordero, M. M.; Houser, J. J.; Wesdemiotis, C. Anal. Chem. 1993, 65, 1594) pathways. General considerations indicate that under low-energy collision conditions the majority of the sequence ions of protonated oligopeptides are formed on the b(x)-y(z) pathways which are energetically, kinetically, and entropically accessible. This is due to the facts that (1).the corresponding reactive configurations (amide N protonated species) can easily be formed during ion excitation, (2). most of the protonated nitrogens are stabilized by nearby amide oxygens making the spatial arrangement of the two amide bonds (the protonated and its N-terminal neighbor) involved in oxazolone formation entropically favored. On the other hand, formation of y ions on the diketopiperazine pathways is either kinetically or energetically or entropically controlled. The energetic control is due to the significant ring strain of small cyclic peptides that are co-formed with y ions (truncated protonated peptides) similar in size to the original peptide. The entropy control precludes formation of y ions much smaller than the original peptide since the attacking N-terminal amino group can rarely get close to the protonated amide bond buried by amide oxygens. Modeling the b(x)-y(z) pathways of protonated pentaalanine leads for the first time to semi-quantitative understanding of the tandem mass spectra of a protonated oligopeptide. Both the amide nitrogen protonated structures (reactive configurations for the amide bond cleavage) and the corresponding b(x)-y(z) transition structures are energetically more favored if protonation occurs closer to the C-terminus, e.g., considering these points the Ala(4)-Ala(5) amide bond is more favored than Ala(3)-Ala(4), and Ala(3)-Ala(4) is more favored than Ala(2)-Ala(3). This fact explains the increasing ion abundances observed for the b(2)/y(3), b(3)/y(2), and b(4)/y(1) ion pairs in the metastable ion and low-energy collision induced mass spectra (Yalcin, T.; Csizmadia, I. G.; Peterson, M. B.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1996, 7, 233) of protonated pentaalanine. A linear free-energy relationship is used to approximate the ratio of the b(x) and y(z) ions on the particular b(x)-y(z) pathways. Applying the necessary proton affinities such considerations satisfactorily explain for example dominance of the b(4) ion over y(1) and the similar b(3) and y(2) ion intensities observed for the metastable ion and low-energy collision induced mass spectra.

Amides↗
PubMed2000

Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels.

Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons.

Biophysical Phenomena↗
PubMed2002

Proton transport via the membrane surface.

Some proton pumps, such as cytochrome c oxidase (C(c)O), translocate protons across biological membranes at a rate that considerably exceeds the rate of proton transport to the entrance of the proton-conducting channel via bulk diffusion. This effect is usually ascribed to a proton-collecting antenna surrounding the channel entrance. In this paper, we consider a realistic phenomenological model of such an antenna. In our model, a homogeneous membrane surface, which can mediate proton diffusion toward the channel entrance, is populated with protolytic groups that are in dynamic equilibrium with the solution. Equations that describe coupled surface-bulk proton diffusion are derived and analyzed. A general expression for the rate constant of proton transport via such a coupled surface-bulk diffusion mechanism is obtained. A rigorous criterion is formulated of when proton diffusion along the surface enhances the transport. The enhancement factor is found to depend on the ratio of the surface and bulk diffusional constants, pK(a) values of surface protolytic groups, and their concentration. A capture radius for a proton on the surface and an effective size of the antenna are found. The theory also predicts the effective distance that a proton can migrate on the membrane surface between a source (such as CcO) and a sink (such as ATP synthase) without fully equilibrating with the bulk. In pure aqueous solutions, protons can travel over long distances (microns). In buffered solutions, the travel distance is much shorter (nanometers); still the enhancement effect of the surface diffusion on the proton flow to a target on the surface can be tens to hundreds at physiological buffer concentrations. These results are discussed in a general context of chemiosmotic theory.

Biophysical Phenomena↗
PubMed1988

Theory of relaxation of mobile water protons induced by protein NH moieties, with application to rat heart muscle and calf lens homogenates.

Kimmich and co-workers (cf., Winter, F., and R. Kimmich. 1982. Biochim. Biophys. Acta. 719:292-298) discovered peaks in the magnetic field-dependent longitudinal relaxation rate (1/T1) of water protons of muscle tissue, cells, and dehydrated protein in the field range 0.5-5 MHz (proton Larmor frequency), and argued that the peaks resulted from cross relaxation associated with quadrupolar splittings of the 14N nuclei of protein NH groups. More recently, analogous peaks were found in homogenates of calf eye lens (Beaulieu, C.F., J.I. Clark, R.D. Brown III, M. Spiller, and S. H. Koenig, 1987. Abstr. Soc. Magn. Res. Med., 6th, New York. 598-599), which are essentially concentrated protein solutions, and were measured with sufficient precision to allow resolution of the relaxation spectra into several peaks and the intrinsic linewidths to be determined. Here, we analyze these relaxation spectra, as well as earlier data on rat heart (Koenig, S. H., R. D. Brown III, D. Adams, D. Emerson, and C. G. Harrison. 1984. Invest. Radiol. 19:76-81) in some detail, and suggest a specific pathway for the cross relaxation to which we apply the theory of relaxation quantitatively. The view that emerges is that, at fields such that the proton Zeeman energy of the NH protons matches an 14N quadrupolar splitting, relaxation of these protons is by cross relaxation to the 14N nuclei which in turn transfer excess energy to the protein. The correlation time for the NH proton interaction is the T2 of the 14N nuclei, approximately 10(-6) s, whereas T1 of the NH protons is approximately 1.25 ms. At these energy level crossings, the NH protons become relaxation sinks for protons of rapidly exchanging (-3 x 109 s-1) water molecules hydrogen bonded to the same backbone carbonyl oxygens as the NH protons. The lifetime of this hydrogen bond (-3 x 10-10 s) then becomes the correlation time for the water proton-NH proton interaction which, though short, is much longer than the analogous correlation time (-5 x 10-12 s) in pure water; the enhanced interaction results in peaks in the field-dependent 1/ T, of the solvent protons. There are few data on the lifetime of such bonds, but the results here conform with the recent considerations of Bennett, H. F., R. D. Brown III, S. H. Koenig, and H. M. Swartz. 1987. Magn. Reson. Med. 4:93-111, regarding hydrogen bond lifetimes for water molecules bound to macromolecules. The recent precise field-dependent relaxation data, here combined with both a quantitative theory and the fact that the magnitude of the 14N peaks is very concentration sensitive, allow, at least for lens proteins, a study of protein-protein interactions difficult to investigate by other methods.

Amines↗
PubMed1996

Disruption of the active site solvent network in carbonic anhydrase II decreases the efficiency of proton transfer.

The importance of maintaining the active site water network for efficient proton transfer was investigated by substituting amino acids of varying size at position 65 in carbonic anhydrase II (including four amino acids found in other CA isozymes, F, L, S, and T, and two amino acids that do not occur naturally at position 65, G and H) and measuring the rate constants for the proton transfer reactions in the variant carbonic anhydrases. Intramolecular proton transfer between zinc-bound water and H64 is significantly inhibited by the introduction of bulky residues at position 65; kcat for CO2 hydration decreases up to 26-fold, comparable to the observed decrease in intramolecular proton transfer caused by removal of H64 [Tu, C., Silverman, D. N., Forsman, C., Jonsson, B.-H., & Lindskog, S. (1989) Biochemistry 28, 7913-7918]. Intermolecular proton transfer between protonated H64 and external buffer is also inhibited, although to a lesser degree. Furthermore, an alternative proton transfer pathway, consisting of an active site solvent-mediated proton transfer from zinc-water to imidazole buffer, is inhibited in the A65F, A65L, and A65H CAII variants. Therefore, the active solvent bridge between zinc-bound water and H64 is disrupted by substitutions at position 65. The inhibition of proton transfer reactions correlates with the disruption of the crystallographically observed solvent network in the CA active site and rotation of the proton acceptor, H64 [Scolnick, L. R., & Christianson, D. W. (1996) Biochemistry 35, 16429-16434], suggesting that this solvent network, including water molecules 292, 264, and 369, or a structurally related network, forms the proton transfer pathway in CAII for both intramolecular proton transfer and stimulation of proton transfer in imidazole buffers.

Binding Sites↗
PubMed2005

Proton exit channels in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal transmembrane enzyme of the respiratory electron transport chain in aerobic cells. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping, a process which results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping and proton exit pathways remain unknown. In our previous work, the continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine cytochrome c oxidase for different redox states of the enzyme. A possible mechanism of oxygen reduction and proton pumping via His291 was proposed. In this paper, using electrostatic calculations, we examine the proton exit pathways in the enzyme. By monitoring the changes of the protonation states, proton affinities, and energies of electrostatic interactions between the titratable groups in different redox states of CcO, we identified the clusters of strongly interacting residues. Using these data, we detected four possible proton exit points on the periplasmic side of the membrane (Lys171B/Asp173B, His24B/Asp25B, Asp51, and Asp300). We then were able to trace the proton exit pathways and to evaluate the energy profiles along the paths. On the basis of energetic considerations and the conservation of the residues in a protein sequence, the most likely exit pathway is one via the Lys171B/Asp173B site. The obtained results are fully consistent with our His291 model of proton pumping, and provide a rationale for the absence of proton leaking in CcO between the pumping strokes.

Animals↗
PubMed2006

Proton exchanges between phenols and ammonia or amines: a computational study.

Density functional theory calculations at the B3LYP/6-31+G(d,p) level of theory have been performed to explore proton exchanges between phenols and ammonia or amines, which can be used to account for previous NMR experiments. For the parent phenol-NH(3) system, a transition state with a symmetric phenolate-NH(4)(+)-like structure, which lies about 35 kcal mol(-1) in energy above the hydrogen-bonded complex, has been successfully located. An intrinsic reaction coordinate (IRC) analysis indicates that the proton exchange is a concerted process, which can be roughly divided into four continuous subprocesses. A series of para-substituted phenol-NH(3) systems have been considered to investigate the substituent effect. Whereas introduction of an electron-withdrawing group on the phenol appreciably reduces the barrier, an opposite effect is observed for an electron-donating group. Moreover, it has been disclosed that there exists a good linear correlation between the activation barriers and the interaction energies between the phenols and NH(3), indicating the important role of proton transfer (or hydrogen bonding) in determining the proton exchange. Also considered are the proton exchanges between phenol and amines and those for some sterically hindered systems. The results show that the phenol tends to exchange hydrogen with the amines, preferably the secondary amines, and that the steric effect is favorable for the proton exchange, which imply that, as the IRC analysis suggested, besides the proton transfer, the flip of the ammonium-like moiety may play a significant role in the course of proton exchange. For all of these systems, we investigated the solvent effects and found that the barrier heights of proton exchange decrease remarkably as compared to those in a vacuum due to the ion pair feature of the transition state. Finally, we explored the phenol radical cation-NH(3) system; the barrierless proton transfer and remarkably low barrier (5.2 kcal mol(-1)) of proton exchange provide further evidence for the importance of proton transfer in the proton exchange.

Amines↗
PubMed1992

Proton-induced sodium current in freshly dissociated hypothalamic neurones of the rat.

1. The proton-gated current was investigated in freshly dissociated ventromedial hypothalamic (VMH) neurones from 4-week-old Wistar rats, under whole-cell configuration by the use of the 'concentration-clamp' technique which combines intracellular perfusion with the rapid exchange of external solution within 1-2 ms under a single-electrode voltage-clamp condition. 2. The proton-gated current increased in a sigmoidal fashion as extracellular pH (pHo) decreased. In external solution containing 2 mM-Ca2+, the threshold of current activation was at pHo 6.5, and the maximum response appeared at pHo 4.1-3.9. The dissociation constant (Kd) and Hill coefficient were 10(-4.9) M (pHo = 4.9) and 1.5 respectively. 3. Decreasing extracellular Na+ concentration reduced the proton-gated current. The current reversed direction at the Na+ equilibrium potential (ENa), indicating that it was carried by Na+. 4. The activation phase kinetics of proton-induced current was single exponential. The time constant of activation (tau a) did not have a potential dependence but decreased slightly by decreasing pHo. The inactivation phase kinetics was two-exponential. The time constant of inactivation (tau i) consisted of fast and slow components (tau if and tau is, respectively). Like tau a, both tau if and tau is did not have any potential dependence, but they slightly increased with decreasing pHo. 5. The steady-state inactivation curve, constructed by decreasing pHo from various conditioning pHos to 4.1, revealed that the proton-induced current had a half-maximum inactivation at pHo 6.2. 6. The proton-induced current was suppressed as the extracellular Ca2+ concentration ([Ca2+]o) increased from almost free (0.01 mM) to 80 mM. Increasing [Ca2+]o increased tau a, but slightly decreased both tau if and tau is. 7. Recovery of proton-induced current from complete inactivation of proton-induced current depended on the degree of pHo change. A bigger change in pHo induced faster recovery than a smaller change. 8. External divalent cations inhibited the proton-induced current, and the inhibitory potency was in the order of Mn2+ greater than Co2+ greater than Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. 9. Tetrodotoxin (TTX) at relatively low concentration (less than 10(-7) M) did not inhibit the peak amplitude of the proton-induced current, but at a higher concentration (10(-6) M) it slightly inhibited the peak amplitude of the current and accelerated the inactivation process. Scorpion toxin markedly increased the peak amplitude of the proton-induced current and prolonged the inactivation phase. The tau is was also increased by scorpion toxin in a concentration-dependent manner. Veratridine had no effect on the proton-induced current. 10. The membrane properties of the proton-operated channel were similar to those of the voltage-gated Na+ channel rather than the Ca2+ channel.

Animals↗
PubMed1988

Protons in the thylakoid membrane-sequestered domains can directly pass through the coupling factor during ATP synthesis in flashing light.

Thylakoid membranes contain sequestered domains in which protons are held in a metastable state out of equilibrium with those in the inner (lumen) or the outer aqueous bulk phases unless the membranes are made leaky by, for instance, the addition of uncouplers. Previously, it has not been clear whether such sequestered domain protons are: 1) directly on a localized pathway into the CF0-CF1; 2) on an ultimately delocalized pathway comprising domains----lumen----CF0-CF1; or 3) perhaps not importantly involved in any way with proton gradient-linked ATP formation. Recent developments now permit a test of the above possibilities. The test for the possible mode of involvement of domain protons in energy coupling utilized single turnover flashes to energize electric field-driven ATP formation, as influenced by both the proton depletion level of the sequestered domains and whether the thylakoids were prepared in a way to show either a localized or delocalized proton gradient response in ATP formation (this response is reversibly controlled by incubation in low (localized coupling mode) or high KCl (delocalized mode) incubations (Beard, W. A., and Dilley, R. A. (1986) FEBS Lett. 201, 57-62; Chiang, G. and Dilley, R. A. (1987) Biochemistry 26, 4911-1916). Using thylakoids showing delocalized coupling responses, there was no influence of the reversible depletion of the sequestered domain buffering pool on the delta psi-driven ATP formation onset lag. Lumenal protons rather than domain protons in that case appear to be the first to be driven through the CF0-CF1 complex by the delta psi field. However, using thylakoids prepared to be in the localized gradient coupling mode, protons in the domains, rather than those in the lumen, are the first to pass through the CF0-CF1 complex in the onset of energization. We conclude that the sequestered domain protons, in the latter case only, are obligatorily in the main proton diffusion pathway for energization of ATP formation and are the first protons (rather than lumen protons) driven through the CF1-CF0 complex by a delta psi field.

Adenosine Triphosphate↗
PubMed1983

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT).

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1977

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.

Adenosine Diphosphate↗
PubMed2005

The effect of octahedral tilting on proton binding sites and transition states in pseudo-cubic perovskite oxides.

Proton conducting oxide ceramics have shown potential for use in fuel cell technologies. Understanding the energy pathways for proton conduction could help us design more efficient fuel cell materials. This paper describes how octahedral tilting affects the relative energies of proton binding sites, transition states, and conduction pathways in cubic and pseudo-cubic perovskites. First, the structure for cubic and pseudo-cubic forms of BaTiO(3), BaZrO(3), CaTiO(3), and CaZrO(3), is found. Even when cubic symmetry is enforced, CaTiO(3), and CaZrO(3) exhibit octahedral tilting distortions characteristic of orthorhombic phases while BaTiO(3) and BaZrO(3) remain undistorted. Octahedral tilting gives rise to proton binding sites facilitating inter- and intra-octahedral proton transfer while the proton binding sites of undistorted perovskites facilitate only intra-octahedral proton transfer. The nudged elastic band method is used to find minimum energy paths between the proton binding sites. As distortions increase, inter-octahedral proton transfer barriers decrease while intra-octahedral proton transfer barriers increase. Concurrently, rotational barriers from oxygens facilitating inter-octahedral proton transfer increase while rotational barriers from oxygens facilitating intra-octahedral proton transfer decrease. Intra-octahedral transfer is the rate-limiting step to the lowest energy extended proton conduction pathway in all the perovskites considered.

Journal Article↗