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PubMed1984

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A.

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.

Concanavalin A↗

PubMed2004

A proton-led model of fast calcium waves.

Fast (10-30 microm/s) calcium waves can be propagated through all nucleated eukaryotic cells that have been tested as well as certain cell-free extracts. In a widely used model, they are propagated by a reaction-diffusion cycle in which calcium ions diffuse along the outside of endoplasmic reticula and induce their own release from calsequestrin or calreticulin molecules stored within the reticulum's lumen. Here we propose a new tandem wave model in which they are also propagated by a reaction-diffusion cycle within a reticulum's lumen. In this cycle, increases in luminal [H(+)] induce proton release from luminal calsequestrin or calreticulin. The released protons diffuse ahead to where they release more protons from these luminal storage proteins. What might be called proton induced proton release. They also raise luminal electropositivity. The resultant luminal waves are coordinated with extrareticular ones by movements of calcium and hydrogen ions through the reticular membrane. This model makes five testable predictions which include the autorelease of protons in solutions of calsequestrins or calreticulins as well as waves of increased [H(+)], of increased [Ca(2+)] and of more positive voltage within the reticula of whole cells. Moreover, under some conditions, such luminal waves should cross regions without cytosolic ones.

Animals↗

PubMed2006

The mechanism of the protonation of metal (hydr)oxides in aqueous solutions studied for various interfacial/surface ionization models and physicochemical parameters: a critical review and a novel approach.

The mechanism of the protonation of solid metal (hydr)oxides in aqueous media was investigated using simulation and experimental work. It was found that the apparent acidity/basicity of each kind of surface sites of metal (hydr)oxides in aqueous suspensions is strongly influenced by the overall surface charge of the (hydr)oxide and thus by the electrical potential smeared out at the interfacial region. Depending on its sign this increases or decreases the hydrogen ion concentration on the surface, thus promoting or hindering protonation. This is manifested by the shifts of the protonation peaks of the various kinds of sites with respect to the -pK values of the corresponding intrinsic protonation constants and the appearance of an extra peak in the d[H+cons,surf]/dpH vs. pH curves. Potentiometric titrations experiments performed for four technologically important oxides showed that the proposed protonation mechanism describes indeed the protonation of polycrystalline (hydr)oxides in aqueous media.

Journal Article↗

PubMed2006

The role of the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) in proton sensitivity of subpopulations of primary nociceptive neurons in rats and mice.

A local elevation of H+-ion concentrations often occurs in inflammation and usually evokes pain by excitation of primary nociceptive neurons. Expression patterns and functional properties of the capsaicin receptor and acid-sensing ion channels suggest that they may be the main molecular substrates underlying this proton sensitivity. Here, we asked how the capsaicin receptor TRPV1 and acid-sensing ion channels (ASICS) contribute to the proton response in subpopulations of nociceptive neurons from adult rats and mice (wildtype C57/Bl6, Balb/C and TRPV1-null). In cultured dorsal root ganglion neurons, whole cell patch clamp recordings showed that the majority of capsaicin-sensitive rat dorsal root ganglion neurons displayed large proton-evoked inward currents with transient ASIC-like properties. In contrast, the prevalence of ASIC-like currents was smaller in both mouse wildtype strains and more frequent in capsaicin-insensitive neurons. Transient ASIC-like currents were more frequent in both species among isolectin B4-negative neurons. A significantly reduced proton response was observed for dissociated dorsal root ganglion neurons in TRPV1 deficient mice. Unmyelinated, but not thin myelinated nociceptors recorded extracellularly from TRPV1-null mutants showed a profound reduction of proton sensitivity. Together these findings indicate that there are significant differences between rat and mouse in the contribution of TRPV1 and ASIC subunits to proton sensitivity of sensory neurons. In both species ASIC subunits are more prevalent in the isolectin B4-negative neurons, some of which may represent thin myelinated nociceptors. However, the main acid-sensor in isolectin B4-positive and isolectin B4-negative unmyelinated nociceptors in mice is TRPV1.

Acid Sensing Ion Channels↗

PubMed2003

Beneficial effect of polyethylene glycol in lung preservation: early evaluation by proton nuclear magnetic resonance spectroscopy.

BACKGROUND: Proton nuclear magnetic resonance spectroscopy can be used to measure organic molecules in biological fluids. In this study, proton nuclear magnetic resonance spectroscopy of bronchoalveolar lavage was assessed to detect cellular damage in lung transplants. Also we evaluated a polyethylene glycol solution in lung preservation. METHODS: An isolated perfused and working pig lung was used to assess initial pulmonary function after in situ cold flush and cold storage for 6 hours in three preservation solutions: (1) Euro-Collins solution, (2) University of Wisconsin solution, and (3) low potassium solution with polyethylene glycol (PEG). Pulmonary vascular resistance and partial pressure of arterial oxygen were measured during reperfusion. Bronchoalveolar lavage was studied by proton nuclear magnetic resonance spectroscopy and a histologic study of the lungs was done at the harvest after ischemia and after reperfusion. RESULTS: Partial pressure of arterial oxygen and pulmonary vascular resistance were significantly better in PEG compared with Euro-Collins solution (p = 0.011). Interstitial edema was significantly higher in Euro-Collins solution (2.4 +/- 0.24; p = 0.02) and University of Wisconsin solution (2.7 +/- 0.20; p = 0.0003) than PEG (2 +/- 0.16). Mitochondria scale was better in PEG (8.1 +/- 0.46) than in Euro-Collins solution (6.2 +/- 0.37; p = 0.0001) or University of Wisconsin solution (5.6 +/- 1.36; p = 0.0046). In bronchoalveolar lavage proton nuclear magnetic resonance spectroscopy spectra, lactate, pyruvate, citrate, and acetate were only detected after reperfusion, with a significantly reduced production of acetate in PEG. Pyruvate was reduced at the limit of significance in PEG versus University of Wisconsin solution. CONCLUSIONS: Proton nuclear magnetic resonance spectroscopy seems to be a simple and suitable method for assessment of early injury to the lung transplant. In this experimental study, PEG preserved the lung better than University of Wisconsin solution and Euro-Collins solution in both the proton nuclear magnetic resonance spectroscopy study as well as the physiologic study.

Animals↗

PubMed1998

Domains, specific residues and conformational states involved in hydride ion transfer and proton pumping by nicotinamide nucleotide transhydrogenase from Escherichia coli.

Nicotinamide nucleotide transhydrogenase constitutes a proton pump which links the NAD(H) and NADP(H) pools in the cell by catalyzing a reversible reduction of NADP+ by NADH. The recent cloning and characterization of several proton-pumping transhydrogenases show that they share a number of features. They are composed of three domains, i.e., the hydrophilic domains I and III containing the NAD(H)- and NADP(H)-binding sites, respectively, and domain II containing the transmembrane and proton-conducting region. When expressed separately, the two hydrophilic domains interact directly and catalyze hydride transfer reactions similar to those catalyzed by the wild-type enzyme. An extensive mutagenesis program has established several amino acid residues as important for both catalysis and proton pumping. Conformational changes mediating the redox-driven proton pumping by the enzyme are being characterized. With the cloned, well-characterized and easily accessible transhydrogenases from E. coli and Rhodospirillum rubrum at hand, the overall aim of the transhydrogenase research, the understanding of the conformationally driven proton pumping mechanism, is within reach.

Binding Sites↗

PubMed1999

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H+/e- ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b6f complex. In order to investigate the H+/e- ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H+/e- ratio occurs with either acceptor. H+/e- ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b6f complex, giving rise to a proton slip reaction at high transmembrane DeltapH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H+/e- ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO2 reduction are discussed.

Journal Article↗

PubMed1999

The proton/sodium antiporter (exchanger) in the oocyte membrane of Dysdercus intermedius is electrogenic (2H(+)/Na(+)) and causes perioocytic proton accumulation.

Vitellogenic follicles of Dysdercus intermedius (Heteroptera: Pyrrhocoridae) were treated with sodium azide (NaN(3)) or 2,4 dinitrophenol (DNP) in order to load the ooplasm with protons along their electrochemical gradient. Ooplasmic pH (pH(OOC)) was recorded using proton-specific microelectrodes. Treatment for six min with 0.5 mM of DNP (dissolved in physiological saline solution; PSS) resulted in acidification of the ooplasm from 7.41+/-0.05 in PSS to pH(OOC(DNP))=7.09+/-0.04. Immersing follicles in PSS after DNP treatment resulted in reactivation of a proton/sodium antiporter and recovery of the initial pH(OOC). Additionally, the proton-specific microelectrodes were placed at a distance of approximately 10 &mgr;m from the surface of the vitellogenic follicle. The extracellular pH (pH(EX)) was measured before (pH(EX(PSS))), during (pH(EX(DNP))) and after (pH(EX(PSS))) DNP treatment. Along the lateral surface of the follicle, the recorded pH(EX(PSS)) was initially 6.79+/-0.02, similar to the pH of the medium (pH(MED)=6.80; recorded at a distance of 300 &mgr;m from the surface of the follicle) and higher than the pH(EX(PSS)) of 6.52+/-0.03 measured in the interfollicular constriction between individual vitellogenic follicles (interfollicular region). During DNP treatment, values changed to 6.80+/-0.03 in the constriction and 6.80+/-0.01 along the lateral surface. After removal of DNP the initial control pH values were reestablished. These extrafollicular H(+) distributions fit into a model of extrafollicular currents reported earlier for D. intermedius.Proton distribution between the ooplasm and the medium was also affected in the presence of 5 mM NaN(3), resulting in a drop in ooplasmic pH from 7.40+/-0.05 down to pH(OOC(NaN3))=7.07+/-0.03. Changes in cytosolic proton activities after DNP or NaN(3) treatment were evidenced by monitoring both the increase in ooplasmic pH (DeltapH) and, simultaneously, the change in the resting potential (DeltaEm). Recovery of the ooplasmic pH depended on the transfer of approximately 6x10(9) H(+)/oocyte (after DNP treatment) or approximately 3x10(9) H(+)/oocyte (after NaN(3) treatment), whereas recovery of Em by charging the capacitance of the oocyte membrane could be attributed to a net efflux of approximately 3x10(9) H(+)/oocyte (after DNP treatment) or approximately 1.7x10(9) H(+)/oocyte (after NaN(3) treatment). In the light of previous reports on the monensin-sensitive proton/sodium antiporter (external Na(+) for ooplasmic H(+)), the operating efficiency of this antiporter is 2H(+)/Na(+).VITELLOGENESIS DURING AND AFTER DNP TREATMENT WAS DEMONSTRATED BY THE ACCUMULATION OF FLUORESCENCE LABELLED HEMOLYMPH PROTEINS IN YOLK SPHERES IN THE CORTEX OF THE OOCYTE: vitellogenesis came to a halt in PSS containing DNP when the ooplasm was acidified and no H(+) accumulation around the follicle was detectable. Vitellogenesis stopped under the condition of DNP(MED)=0.5 mM, but resumed again by exchanging the medium for PSS without DNP. Simultaneously with the appearance of the regular pH(OOC(PSS))=7.40+/-0.03 (efflux of H(+) out of the ooplasm), extrafollicular proton accumulation by H(+) influx into the constriction reappeared within 10 minutes. The results obtained with proton-specific microelectrodes and the in vitro assay to detect vitellogenesis indicate that electrogenic H(+) extrusion out of the ooplasm plays an important role in both maintaining the ooplasmic pH 0.6 units above pH(MED)=6.8 and in the generation of the external current pattern. A model is discussed explaining the acidification of endosomes as a prerequisite for endosomal processing leading to yolk spheres.

Journal Article↗

PubMed2002

Critical appraisal of treatment techniques based on conventional photon beams, intensity modulated photon beams and proton beams for therapy of intact breast.

PURPOSE: To analyse different treatment techniques with conventional photon beams, intensity modulated photon beams, and proton beams for intact breast irradiation for patients in whom conventional irradiation would cause potentially dangerous lung irradiation. MATERIALS AND METHODS: Five breast cancer patients with highly concave breast tissue volume around the lung were considered at planning level in order to assess the suitability of different irradiation techniques. Three-dimensional dose distributions for conventional two-field tangential photon treatment, two-field intensity modulated radiotherapy (IMRT), three-field non-IMRT, three-field IMRT, and single-field proton treatment were investigated, aiming at assessing the possibility to reduce lung irradiation below risk levels. Analysis of dose-volume histograms and related physical and biological parameters (significant minimum, maximum and mean doses, conformity indexes and equivalent uniform dose (EUD)) for planned target volume (PTV) and lung was carried out. Dose plans were compared with the conventional two-field tangential photon technique. RESULTS: PTV coverage was comparable for non-IMRT and IMRT techniques (EUD from 47.1 to 49.4 Gy), and improved with single-field proton treatment (EUD=49.8 Gy). Lung irradiation was reduced, in terms of mean dose, with three-field (9.5 Gy) and proton technique (3.5 Gy), with respect to the conventional two-field treatment (12.9 Gy); also a reduction of the lung volume irradiated at high doses was observed. Better results could be achieved with protons. In addition, cardiac irradiation was also reduced with those techniques. CONCLUSIONS: Geometrically difficult breast cancer patients could be irradiated with a three-field non-IMRT technique thus reducing the dose to the lung which is proposed as standard for this category of patients. Intensity modulated techniques were only marginally more successful than the corresponding non-IMRT treatments, while protons offer excellent results.

Breast↗

PubMed1998

An experimental methodology for measuring volume changes in proton transfer reactions in aqueous solutions.

A fast perturbation in proton concentration can be induced in aqueous solution using a pulsed ultraviolet laser and suitable photolabile compounds which, upon photoexcitation, irreversibly release protons. The volume change and the rate constant for the reaction of the photodetached protons with proton-accepting groups in solution can be monitored using time resolved photoacoustics. A typical proton concentration jump of 1 microM can be obtained with a 200-microJ laser pulse at 308 nm. Reaction dynamics from 20 ns to 5 micros can be easily followed. The methodology we establish represents a direct, time-resolved measurement of the reaction volume in proton transfer processes and an extension to the nanosecond-microsecond range of traditional relaxation techniques, such as stopped-flow. We report example applications to reactions involving simple molecules and polypeptides.

Journal Article↗

PubMed1999

T2* and proton density measurement of normal human lung parenchyma using submillisecond echo time gradient echo magnetic resonance imaging.

OBJECTIVE: To obtain T2* and proton density measurements of normal human lung parenchyma in vivo using submillisecond echo time (TE) gradient echo (GRE) magnetic resonance (MR) imaging. MATERIALS AND METHODS: Six normal volunteers were scanned using a 1.5-T system equipped with a prototype enhanced gradient (GE Signa, Waukausha, WI). Images were obtained during breath-holding with acquisition times of 7-16 s. Multiple TEs ranging from 0.7 to 2.5 ms were tested. Linear regression was performed on the logarithmic plots of signal intensity versus TE, yielding measurements of T2* and proton density relative to chest wall muscle. Measurements in supine and prone position were compared, and effects of the level of lung inflation on lung signal were also evaluated. RESULTS: The signal from the lung parenchyma diminished exponentially with prolongation of TE. The measured T2* in six normal volunteers ranged from 0.89 to 2.18 ms (1.43 +/- 0.41 ms, mean +/- S.D.). The measured relative proton density values ranged between 0.21 and 0.45 (0.29 +/- 0.08, mean +/- S.D.). Calculated T2* values of 1.46 +/- 0.50, 1.01 +/- 0.29 and 1.52 +/- 0.18 ms, and calculated relative proton densities of 0.20 +/- 0.03, 0.32 +/- 0.13 and 0.35 +/- 0.10 were obtained from the anterior, middle and posterior portions of the supine right lung, respectively. The anterior-posterior proton density gradient was reversed in the prone position. There was a pronounced increase in signal from lung parenchyma at maximum expiration compared with maximum inspiration. The ultrashort TE GRE technique yielded images demonstrating signal from lung parenchyma with minimal motion-induced noise. CONCLUSION: Quantitative in vivo measurements of lung T2* and relative proton density in conjunction with high-signal parenchymal images can be obtained using a set of very rapid breath-hold images with a recently developed ultrashort TE GRE sequence.

Artifacts↗

PubMed2000

Protonation in electrospray mass spectrometry: wrong-way-round or right-way-round?

The term "wrong-way-round ionization" has been used in studies of electrospray ionization to describe the observation of protonated or deprotonated ions when sampling strongly basic or acidic solutions (respectively) where such ions are not expected to exist in appreciable concentrations in solution. Study of the dependence of ionization of the weak base caffeine on the electrospray capillary potential reveals three distinct contributors to wrong-way-round ionization. At near-neutral pH in solutions of low ionic strength, protonation of caffeine results from the surface enrichment of electrolytically produced protons in the surface layer of the droplets from which ions are desorbed. For solutions made strongly basic with ammonia, gas-phase proton transfer from ammonium ions can create protonated caffeine. These two mechanisms have been discussed previously elsewhere. For solutions of high ionic strength at neutral or high pH, the data suggest that discharge-induced ionization is responsible for the production of protonated caffeine. This mechanism probably accounts for some of the wrong-way-round ionization reported elsewhere.

Journal Article↗

PubMed2005

Using an oxide nanoarchitecture to make or break a proton wire.

We report that long-range proton diffusion (>0.3 mm) is generated in monolithic ultraporous manganese oxide nanoarchitectures upon exposure to gas-phase water. The sol-gel-derived ambigel nanoarchitectures, with bicontinuous networks of covalently bonded nanoscale solid and through-connected mesopores, exhibit conductometric sensitivity to humidity as established by impedance spectroscopy. The spectra contain a Warburg feature from which the concentration and diffusion length of the protonic charge carriers are determined. Water adsorbs conformally onto the architecture's continuous solid network in equilibrium with atmospheric humidity to create a continuous water sheath that acts as a 3-D proton wire. As a result, monolithic manganese oxide ambigels exhibit an equilibrium conductometric response to humidity that is 14 times greater than that of previous reports for electrolytic manganese oxide. A packed bed of 1-10-microm ambigel particulates in physical contact with one another, each with the same nanoscale morphology as the monolithic nanoarchitecture, also support long-range proton diffusion; however, the sensitivity to humidity is four times lower than the monolithic form due to restricted proton transport between adjacent particulates. Films composed of 0.3-12-microm ambigel particulates supported on interdigitated array electrodes with 20-microm electrode spacing express finite-diffusion behavior due to the short distance between the contact electrodes and have a conductometric sensitivity to humidity comparable to electrolytic MnO2 and 17 times lower than the monolithic ambigel. These results suggest that controlling the nature of the porous and solid phases in a nanoarchitecture provides a mechanism to limit interference from condensed water in conductometric gas-phase sensors. In addition, continuous monolithic architectures should improve electrochemical performance in devices where efficient long-range transport of protons or other ions is critical.

Journal Article↗

PubMed1995

Involvement of histidine-91 of the beta subunit in proton translocation by the pyridine nucleotide transhydrogenase of Escherichia coli.

The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at this residue affected the conformation of the enzyme. Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH. By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with [14C]N,N'-dicyclohexylcarbodiimide (DCCD). Labeling occurred most readily with the enzyme of beta H91K. It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme.

Amino Acid Sequence↗

PubMed1995

Proton transfer by histidine 67 in site-directed mutants of human carbonic anhydrase III.

The ability of a histidine residue at position 67 in human carbonic anhydrase III to transfer protons in the catalytic pathway for the hydration of CO2 was investigated for a series of site-specific mutants. Wild-type carbonic anhydrase III has an arginine at this position with the C alpha of residue 67 about 9.4 A from the zinc. The active-site cavity contains no other residues capable of facile proton transfer. Rate constants for proton transfer from His 67 to the zinc-bound hydroxide were determined from the rate constants for the exchange of 18O between CO2 and water measured by mass spectrometry. A range of values for the pKa of zinc-bound water was achieved by replacement of phenylalanine with leucine and aspartate at position 198 adjacent to the zinc. Application of Marcus rate theory showed that intramolecular proton transfer involving His 67 had an intrinsic energy barrier of 1.3 +/- 0.3 kcal/mol and a thermodynamic work function for a preceding unfavorable equilibrium of 10.9 +/- 0.1 kcal/mol. We previously showed that proton transfer from histidine 64 in carbonic anhydrase III could be described by Marcus rate theory [Silverman, D. N., Tu, C. K., Chen, X., Tanhauser, S. M., Kresge, A. J., & Laipis, P. J. (1993) Biochemistry 32, 10757-10762]. In comparison, proton transfer from His 67 must overcome a more unfavorable preceding equilibrium (a larger work function) that probably represents an energy requirement for proper alignment of donor and acceptor groups plus the intervening hydrogen-bonded water. Once this alignment is achieved, the intrinsic energy barrier appears the same for His 67 or His 64.

Amino Acid Sequence↗

PubMed1993

Proton transfer from Asp-96 to the bacteriorhodopsin Schiff base is caused by a decrease of the pKa of Asp-96 which follows a protein backbone conformational change.

In the bacteriorhodopsin photocycle the transported proton crosses the major part of the hydrophobic barrier during the M to N reaction; in this step the Schiff base near the middle of the protein is reprotonated from D96 located near the cytoplasmic surface. In the recombinant D212N protein at pH > 6, the Schiff base remains protonated throughout the photocycle [Needleman, Chang, Ni, Váró, Fornés, White, & Lanyi (1991) J. Biol. Chem. 266, 11478-11484]. Time-resolved difference spectra in the visible and infrared are described by the kinetic scheme BR-->K<==>L<==>N (-->N')-->BR. As evidenced by the large negative 1742-cm-1 band of the COOH group of the carboxylic acid, deprotonation of D96 in the N state takes place in spite of the absence of the unprotonated Schiff base acceptor group of the M intermediate. Instead of internal proton transfer to the Schiff base, the proton is released to the bulk, and can be detected with the indicator dye pyranine during the accumulation of N'. The D212N/D96N protein has a similar photocycle, but no proton is released. As in wild-type, deprotonation of D96 in the N state is accompanied by a protein backbone conformational change indicated by characteristic amide I and II bands. In D212N the residue D96 can thus deprotonate independent of the Schiff base, but perhaps dependent on the detected protein conformational change. This could occur through increased charge interaction between D96 and R227 and/or increased hydration near D96. We suggest that the proton transfer from D96 to the Schiff base in the wild-type photocycle is driven also by such a decrease in the pKa of D96.

Amino Acid Sequence↗

PubMed1993

Substitution of asparagine for aspartate-135 in subunit I of the cytochrome bo ubiquinol oxidase of Escherichia coli eliminates proton-pumping activity.

The terminal quinol oxidase, cytochrome bo, of Escherichia coli is a member of the large terminal oxidase family, which includes cytochrome aa3-type terminal oxidases from bacteria, plants, and animals. These enzymes conserve energy by linking electron transfer to vectorial proton translocation across mitochondrial or bacterial cell membranes. Site-directed mutagenesis of the five most highly conserved acidic amino acids in subunit I of cytochrome bo was performed to study their role in proton transfer. Mutation of only one of these sites, Asp135, to the corresponding amide, results in a dramatic decrease in proton pumping but with little change in electron-transfer activity. However, the conservative mutation Asp135Glu is active in proton translocation. It is proposed that an acidic residue at position 135 in subunit I may be important to form a functional proton input channel of the proton pump.

Amino Acid Sequence↗

PubMed1992

Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation.

Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.

Base Sequence↗