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PubMed2001

Grotthus-type and diffusive proton transfer in 7-hydroxyquinoline.(NH(3))(n) clusters.

Proton translocation along ammonia wires is investigated in 7-hydroxyquinoline.(NH(3))(n) clusters, both experimentally by laser spectroscopy and theoretically by Hartree-Fock and density functional (DFT) calculations. These clusters serve as realistic finite-size models for proton transfer along a chain of hydrogen-bonded solvent molecules. In the enol tautomer of 7-hydroxyquinoline (7-HQ), the OH group acts as a proton injection site into the (NH(3))(n)cluster. Proton translocation along a chain of three NH(3) molecules within the cluster can take place, followed by reprotonation of 7-HQ at the quinolinic N atom, forming the 7-ketoquinoline tautomer. Exoergic proton transfer from the OH group of 7-HQ to the closest NH(3) molecule within the cluster giving a zwitterion 7-HQ-.(NH(3))(6)H+ (denoted PT-A) occurs at a threshold cluster size of n = 6 in the DFT calculations and at n = 5 or 6 experimentally. Three further locally stable zwitterion clusters denoted PT-B, PT-B', and PT-C, the keto tautomer, and several transition structures along the proton translocation path were characterized theoretically. Grotthus-type proton-hopping mechanisms occur for three of the proton transfer steps, which have low barriers and are exoergic or weakly endoergic. The step with the highest barrier involves a complex proton transfer mechanism, involving structural reorganization and large-scale diffusive motions of the cluster.

Journal Article↗
PubMed2002

Ab initio calculations and mass spectrometric determination of the gas-phase proton affinities of 4,4'-disubstituted 2,2'-bipyridines.

The gas-phase proton affinities of 4,4'-di(R)-2,2'-bipyridines (R: H, Br, Cl, NO(2), Me) were determined by mass spectrometric measurements and by ab initio calculations at the HF/6-31G and MP2/6-31G levels of theory. The energy barriers for rotation about the central C-C bond were also studied computationally. Two minima were found for both unprotonated and protonated species, the global minima being at the trans planar and cis planar conformations, respectively. Local minima for the unprotonated compounds were at the cis nonplanar conformation and for the protonated compounds at the trans nonplanar. Two different proton affinity values were calculated for each compound by employing different conformations for the protonated species. The computational values were in good agreement with the experimental proton affinities. Substituents affect the proton affinity according to their ability to withdraw or to donate electrons, halogen and nitro-substituted bipyridines having a lower proton affinity and methyl-substituted bipyridine having a higher proton affinity than 2,2'-bipyridine itself.

Journal Article↗
PubMed2006

Primary semiclassical kinetic hydrogen isotope effects in identity carbon-to-carbon proton- and hydride-transfer reactions, an ab initio and DFT computational study.

Enthalpies of activation, transition state (ts) geometries, and primary semiclassical (without tunneling) kinetic isotope effects (KIEs) have been calculated for eleven bimolecular identity proton-transfer reactions, four intramolecular proton transfers, four nonidentity proton-transfer reactions, eleven identity hydride transfers, and two 1,2-intramolecular hydride shifts at the HF/6-311+G, MP2/6-311+G, and B3LYP/6-311++G levels. We find the KIEs to be systematically smaller for hydride transfers than for proton transfers. This outcome is not the result of "bent" transition states, although extreme bending can lower the KIE. Rather, it is a consequence of generally greater total bonding in a hydride-transfer ts than in a proton-transfer ts, most prominently manifested as a reduced contribution from the zero-point vibrational energy difference between reactant and transition states (the DeltaZPVE factor) for hydride transfers relative to proton transfers. This and other differences between proton and hydride transfers are rationalized by modeling the central .C...H...C unit of a proton-transfer ts as a 4-electron, 3-center (4-e 3-c) system and the same unit of a hydride-transfer ts as a 2-e 3-c system. Inclusion of tunneling is most likely to magnify the observed differences between proton-transfer and hydride-transfer KIEs, leaving our qualitative conclusions unchanged.

Journal Article↗
PubMed2005

FTIR adsorption studies of H2O and CH3OH in the isostructural H-SSZ-13 and H-SAPO-34: formation of H-bonded adducts and protonated clusters.

The acidity of the isostructural H-SSZ-13 and H-SAPO-34 has been investigated by transmission FTIR spectroscopy using H2O and CH3OH as molecular probes. Interactions between the zeolitic samples and the probe molecules led to perturbations and proton transfers directly related to the acidity of the materials. The entire set of acidic sites in H-SSZ-13 interacts with H2O and CH3OH to give H-bonded adducts or protonated species. H3O+ is not formed in appreciable amounts upon H2O adsorption on H-SSZ-13, but at high coverages H2O generates clusters that have a proton affinity sufficiently high to abstract protons from the zeolite framework. Parallel experiments carried out for H-SAPO-34 showed that the H2O clusters abstract protons from Brønsted sites only to a minor extent. Moving to CH3OH, even if it has a higher proton affinity than H2O and should expectingly experience an easier protonation, proton transfer is totally absent in H-SAPO-34 under our set of conditions. The clear evidence of methanol protonation in H-SSZ-13 definitely states the strong acidic character of this material. When irreversibly adsorbed CH3OH is present in H-SSZ-13, an appreciable amount of (CH3)2O is formed upon heating to 573 K. Compared to its SAPO analogue, the present set of data indisputably points to H-SSZ-13 as the strongest Brønsted acidic material.

Journal Article↗
PubMed2005

Infrared spectra of homogeneous and heterogeneous proton-bound dimers in the gas phase.

Infrared multiphoton dissociation spectra of three homogeneous and two heterogeneous proton-bound dimers were recorded in the gas phase. Comparison of the experimental infrared spectra recorded in the fingerprint region of the proton-bound dimers with spectra predicted by electronic structure calculations shows that all modes which are observed contain motion of the proton oscillating between the two monomers. The O-H-O asymmetric stretch for the homogeneous dimers is shown to occur at around 800 cm-1. As expected, the O-H-O asymmetric stretching modes for the heterogeneous proton-bound dimers are observed to shift to significantly higher energy with respect to those for the homogeneous proton-bound dimers due to the asymmetry of the O-H-O moeity. This shift is shown to be predictable from the difference in proton affinities between the two monomers. Density functional predictions of the infrared spectra based on the harmonic oscillator model are demonstrated to predict the observed spectra of the homogeneous proton-bound dimers with reasonable accuracy. Calculations of the structure and infrared spectrum of protonated diglyme at the B3LYP/6-31+G** level and basis also agree well with an infrared spectrum recorded previously. For both heterogeneous proton-bound dimers, however, the predicted spectra are blue-shifted with respect to experiment.

Dimerization↗
PubMed1984

Cooperative transient trapping of photosystem II protons by the integral membrane portion (CF0) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1).

The ATP-synthase in chloroplasts is built from two blocks, CF0, which is integral to the thylakoid membrane and which serves as a proton channel, and CF1, attached to CF0, which is catalytically active. This study is aimed at understanding proton conduction through CF0. By a mild procedure we extracted <10% of total CF1, predominantly the four-subunit CF1 without the delta subunit. Extracted chloroplasts were excited with short flashes of light and the time course of the transmembrane potential and of the pH changes in both phases was measured spectrophotometrically. Mild extraction of CF1 caused two effects. (i) Up to 50% of the protons rapidly released from water oxidation transiently escaped detection in the thylakoid interior. (ii) The initial extent of the transmembrane potential was decreased by some 10% (20-mus resolution). Protons that were not detected inside appeared in the external phase after having passed the thylakoid membrane. pH titrations of the transient loss of protons produced an extremely sharp transition (near pH 7.5) as if six protons were buffered in a strictly cooperative manner. These effects were reversed upon addition of N,N'-dicyclohexylcarbodiimide, which, among other actions, blocks the proton channel through CF0. We interpret these observations as follows. (i) CF0 incorporates proton binding groups, which can act in a hexacooperative way. These groups are located near the middle of the membrane. (ii) After extraction of CF1, protons produced during water oxidation have very rapid access to these groups, but they pass the full span of the membrane more slowly: buffering precedes conduction through CF0.

Journal Article↗
PubMed2003

Extracellular Ca2+ modulates the effects of protons on gating and conduction properties of the T-type Ca2+ channel alpha1G (CaV3.1).

Since Ca2+ is a major competitor of protons for the modulation of high voltage-activated Ca2+ channels, we have studied the modulation by extracellular Ca2+ of the effects of proton on the T-type Ca2+ channel alpha1G (CaV3.1) expressed in HEK293 cells. At 2 mM extracellular Ca2+ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca2+. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca2+-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca2+. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca2+ and Na+. Mutation of the EEDD pore locus altered the Ca2+-dependent proton effects on channel selectivity and permeation. We conclude that Ca2+ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.

Calcium↗
PubMed1982

Proton-sulfate co-transport: mechanism of H+ and sulfate addition to the chloride transporter of human red blood cells.

Proton and sulfate inhibition of the obligatory chloride-chloride exchange of human erythrocytes was measured at 0 degrees C to determine their mechanism of reaction with the anion transporter. The proton and sulfate that are co-transported by this mechanism at higher temperatures behaved as nontransported inhibitors at 0 degrees C. We analyzed the data in terms of four molecular mechanisms: (1) HSO4- addition to the transporter; (2) ordered addition with the proton first; (3) ordered addition with the sulfate first; (4) random addition to the transporter. The Dixon plots of 1/MCl vs. [SO4] at different proton concentrations were not parallel. Thus protons and sulfate ions were not mutually exclusive inhibitors. The slope of these Dixon plots was independent of pH above 7.0, which indicates that sulfate could bind to the unprotonated carrier and excludes the first two mechanisms. Protons were inhibitors of chloride flux in the absence of sulfate, which indicates that protons could bind to the unloaded carrier and excludes mechanism 3. The KI for sulfate was 4.35 +/0 0.36 mM. The pK for the protonatable group was 5.03 +/- 0.02. The binding of either a proton or sulfate to the carrier decreased the KI of the other by ninefold. The only simple mechanism consistent with the data is a random-ordered mechanism with more transporters loaded with a sulfate than loaded with a proton at the pH and sulfate concentrations of plasma.

Biological Transport, Active↗
PubMed2006

Effects of target plasma electron-electron collisions on correlated motion of fragmented protons.

The objective of the present work is to examined the effects of plasma target electron-electron collisions on H2 + protons traversing it. Specifically, the target is deuterium in a plasma state with temperature Te=10 eV and density n=10(23) cm(-3), and proton velocities are vp=vth, vp=2vth, and vp=3vth, where vth is the electron thermal velocity of the target plasma. Proton interactions with plasma electrons are treated by means of the dielectric formalism. The interactions among close protons through plasma electronic medium are called vicinage forces. It is checked that these forces always screen the Coulomb explosions of the two fragmented protons from the same H2 + ion decreasing their relative distance. They also align the interproton vector along the motion direction, and increase the energy loss of the two protons at early dwell times while for longer times the energy loss tends to the value of two isolated protons. Nevertheless, vicinage forces and effects are modified by the target electron collisions. These collisions enhance the calculated self-stopping and vicinage forces over the collisionless results. Regarding proton correlated motion, when these collisions are included, the interproton vector along the motion direction overaligns at slower proton velocities (vp=vth) and misaligns for faster ones (vp=2vth, vp=3vth). They also contribute to a great extend to increase the energy loss of the fragmented H2 + ion. This later effect is more significant in reducing projectile velocity.

Journal Article↗
PubMed1987

The effect of vanadate on proton-sucrose cotransport in ricinus cotyledons.

The effects of orthovanadate on the uptake of sucrose by Ricinus cotyledons and on sucrose-coupled proton influx were measured in order to gain insight into the relationship to the plasma membrane proton pump. Vanadate had no effect on short-term sucrose uptake. In longterm experiments (>30 min) sucrose uptake was progressively inhibited, but only at high external sucrose concentrations. Vanadate did not affect proton efflux pumping in the absence of sucrose and neither did it change the initial rate of sucrose-coupled proton influx. However, it enhanced the maximal level of sucrose-induced alkalinization of the medium at all sucrose concentrations tested. This is interpreted as an inhibiting effect of vanadate on the proton pump that recycles protons during sucrose-proton cotransport. The sensitivity towards vanadate indicates that this proton pump is an ATPase. A second proton-translocating system, that is insensitive to vanadate, is postulated to function in the absence of sucrose.

Journal Article↗
PubMed1987

Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : A Comparative Study of Ion Effects on DeltapH and DeltaPsi.

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon DeltapH and DeltaPsi revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce DeltaPsi. For tonoplast vesicles, the relative order for this effect was KI > KBr >/= KCl > KClO(3) > K(2)SO(4) while for plasma membrane vesicles, a different order KI > KNO(3) >/= KBr >/= KClO(3) > KCl > K(2)SO(4) was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K(+)/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.

Journal Article↗
PubMed1978

A hydrogen-deuterium exchange study of the amide protons of polymyxin B by nuclear-magnetic-resonance spectroscopy.

1. Proton magnetic resonance spectra at 270 MHz of polymyxin B, a cationic oligopeptide antibiotic, show the influence of the inorganic counteranion present in solution. 2. Hydrogen-deuterium exchange rates for the amide protons are of two types, depending on whether the anion is monovalent or polyvalent. Polyvalent anions catalyse the acid-catalysed reaction more than the monovalent anions. 3. The structure in solution was monitored using the proton signals of the amides, the phenylalanine aromatic protons, and the leucine methyl and gamma-CH protons in several polymyxin salts. The temperature coefficients of the chemical shifts of the N-H protons are used to identify two beta turns in the cyclic ring of polymyxin B. The variation in chemical shift of the N-H protons, the aromatic protons and the leucine protons are correlated with anionic size and electronegativity.

Amino Acid Sequence↗
PubMed1981

Proton-nuclear-magnetic-resonance study on molecular conformations of long neurotoxins. alpha-Bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja.

The 270-MHz proton NMR spectra were analyzed of the long neurotoxins alpha-bungarotoxin from Bungarus multicinctus and Toxin B from Naja naja. The aromatic proton resonances were completely assigned to individual nuclei for alpha-bungarotoxin and in part for toxin B. The pH dependences of proton chemical shifts were analyzed by the nonlinear least-square method, for obtaining pKa values and protonation shifts. The pKa values of Tyr-25, an invariant residue of neurotoxins, are 12.1 for alpha-bungarotoxin and 11.3 for toxin B, suggesting the presence of a strong hydrogen bond involving Tyr-25 in alpha-bungarotoxin. The Trp-29 residues of both toxins show a common titration shift due to the carboxylate group of Asp-31 and a similar structural arrangement of functionally invariant pair of Trp-29 and Asp-31 is implied. From the temperature dependences of the chemical shifts of His-68 and a methyl group of alpha-bungarotoxin, the local structure around His-68 near the tail part is shown to be more flexible than the other part. The six main-chain amide protons of alpha-bungarotoxin exchange most slowly with solvent deuterons and are found by interproton nuclear Overhauser effects to be in the beta-sheet near the aromatic ring of Tyr-25 residue. Hydrogen leads to deuterium exchange rates in 2H2O solution at 37 degrees C were measured of slowly exchanging amide protons of alpha-bungarotoxin, toxin B, and two short neurotoxins, namely cobrotoxin and erabutoxin b. The two long neurotoxins have amide protons with relatively long half-times spanning as long as 10-100 h, but the two short neurotoxins do not have amide protons with half-times longer than 3 h. The distributions of the half-times of amide proton exchange indicate the structural rigidity of neurotoxins in the order, alpha-bungarotoxin greater than toxin B greater than cobrotoxin approximately erabutoxin b, in agreement with the order of neurotoxicity as reported previously by Chicheportiche et al. and by Lee and Chen.

Amino Acid Sequence↗
PubMed1978

Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex.

A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.

Adenosine Triphosphatases↗
PubMed1995

Sodium-proton exchange and primary hypertension. An update.

An enhancement of sodium-proton exchange in blood cells of patients with primary hypertension has been described by various investigators. The present review summarizes some of the most recent findings regarding the enhanced sodium-proton exchanger activity in primary hypertension and discusses the potential mechanisms that may contribute to or explain these findings. Novel evidence has been accumulated on the in vivo regulation of the sodium-proton exchanger in humans, and recent findings suggest that metabolic acidosis, high NaCl intake, and circulating hormones (eg, insulin) can enhance sodium-proton exchanger activity in blood cells. However, the relative roles of such exogenous factors in the stimulation of sodium-proton exchanger activity in primary hypertension remain questionable because enhanced sodium-proton exchanger activity persists in immortalized lymphoblasts from patients with primary hypertension after prolonged cell culture. Therefore, at least in a certain group of hypertensive subjects this abnormality cannot be due to metabolic or hormonal alterations of the "hypertensive" in vivo milieu but appears to be under genetic control. Available evidence strongly argues against intrinsic changes of the sodium-proton exchanger protein itself in primary hypertension, for example, a mutation in the encoding gene. Interestingly, immortalized cells from hypertensive subjects with enhanced sodium-proton exchanger activity display a distinctly enhanced proliferation pattern that appears to be independent of this ion transport. At present we speculate that enhanced sodium-proton exchanger activity and proliferation may represent indicators of a genetically fixed enhanced intracellular signal transduction in primary hypertension that may be caused by an increased activation of pertussis toxin-sensitive G proteins.

Animals↗
PubMed2003

The gas-phase basicity and proton affinity of 1,3,5-cycloheptatriene--energetics, structure and interconversion of dihydrotropylium ions.

The hitherto unknown gas-phase basicity and proton affinity of 1,3,5-cycloheptatriene (CHT) have been determined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. Several independent techniques were used in order to exclude ambiguities due to proton-induced isomerisation of the conjugate cyclic C(7)H(9)(+) ions, [CHT + H](+). The gas-phase basicity obtained by the thermokinetic method, GB(CHT) = 799 +/- 4 kJ mol(-1), was found to be identical, within the limits of experimental error, with the values measured by the equilibrium method starting with protonated reference bases, and with the values resulting from the measurements of the individual forward and reverse rate constants, when corrections were made for the isomerised fraction of the C(7)H(9)(+) population. The experimentally determined gas-phase basicity leads to the proton affinity of cycloheptatriene, PA(CHT) = 833 +/- 4 kJ mol(-1), and the heat of formation of the cyclo-C(7)H(9)(+) ion, deltaH(f)(0)([CHT + H](+)) = 884 +/- 4 kJ mol(-1). Ab initio calculations are in agreement with these experimental values if the 1,2-dihydrotropylium tautomer, [CHT + H((1))](+), generated by protonation of CHT at C-1, is assumed to be the conjugate acid, resulting in PA(CHT) = 825 +/- 2 kJ mol(-1) and deltaH(f)(0)(300)([CHT + H((1))](+)) = 892 +/- 2 kJ mol(-1). However, the calculations indicate that protonation of cycloheptatriene at C-2 gives rise to transannular C-C bond formation, generating protonated norcaradiene [NCD + H](+), a valence tautomer being 19 kJ mol(-1) more stable than [CHT + H((1))](+). The 1,4-dihydrotropylium ion, [CHT + H((3))](+), generated by protonation of CHT at C-3, is 17 kJ mol(-1) less stable than [CHT + H((1))](+). The bicyclic isomer [NCD + H](+) is separated by relatively high barriers, 70 and 66 kJ mol(-1) from the monocyclic isomers, [CHT + H((1))](+) and [CHT + H((3))](+), respectively. Therefore, the initially formed 1,2-dihydrotropylium ion [CHT + H((1))](+) does not rearrange to the bicyclic isomer [NCD + H](+) under mild protonation conditions.

Journal Article↗
PubMed1992

Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the Schiff base.

In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 +/- 0.1 and the apparent pK for protonation of the His85 side chain to be approximately 3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.

Amino Acid Sequence↗
PubMed1986

Identification of a vanadate-sensitive potassium-dependent proton pump from rabbit colon.

Membrane vesicles prepared from rabbit distal colon were used to study colonic transport mechanisms. Differential and sucrose-Ficoll density gradient centrifugation of the mucosal homogenate yielded fractions which supported ATP-dependent proton transport, as measured with the fluorescent weak base acridine orange. Quenching of acridine orange absorbance in light microsomes and microsome-derived density gradient fractions was MgATP-dependent and was reversed with nigericin; these characteristics suggest the presence of one or more ATP-driven proton pumps. Proton transport in the microsomal fraction was inhibited by N-ethylmaleimide more than by orthovanadate, and was dependent on extravesicular chloride. Vesicles in a microsome-derived gradient fraction were inhibited by orthovanadate more than by N-ethylmaleimide. N-ethylmaleimide pretreatment of this gradient fraction uncovered a vesicle population with characteristics similar to the gastric H+,K+ATPase: proton transport was abolished by orthovanadate and the experimental anti-ulcer drug SCH 28080, was enhanced by potassium, and was not affected by chloride. ATP-generated proton gradients in this fraction were not dissipated by the proton ionophore 3,3',4',5-tetrachlorosalicylanilide. We conclude that two ATP-driven proton pumps are present in mucosa from distal rabbit colon; one with characteristics of N-ethylmaleimide-sensitive organelle associated proton pumps, and one similar to the gastric proton-potassium exchanger.

Adenosine Triphosphatases↗