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PubMed2006

pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase. The role of H2O produced at the oxygen-reduction site.

A study is presented on the pH dependence of proton translocation in the oxidative and reductive phases of the catalytic cycle of purified cytochrome c oxidase (COX) from beef heart reconstituted in phospholipid vesicles (COV). Protons were shown to be released from COV both in the oxidative and reductive phases. In the oxidation by O2 of the fully reduced oxidase, the H+/COX ratio for proton release from COV (R --> O transition) decreased from approximately 2.4 at pH 6.5 to approximately 1.8 at pH 8.5. In the direct reduction of the fully oxidized enzyme (O --> R transition), the H+/COX ratio for proton release from COV increased from approximately 0.3 at pH 6.5 to approximately 1.6 at pH 8.5. Anaerobic oxidation by ferricyanide of the fully reduced oxidase, reconstituted in COV or in the soluble case, resulted in H+ release which exhibited, in both cases, an H+/COX ratio of 1.7-1.9 in the pH range 6.5-8.5. This H+ release associated with ferricyanide oxidation of the oxidase, in the absence of oxygen, originates evidently from deprotonation of acidic groups in the enzyme cooperatively linked to the redox state of the metal centers (redox Bohr protons). The additional H+ release (O2 versus ferricyanide oxidation) approaching 1 H+/COX at pH < or = 6.5 is associated with the reduction of O2 by the reduced metal centers. At pH > or = 8.5, this additional proton release takes place in the reductive phase of the catalytic cycle of the oxidase. The H+/COX ratio for proton release from COV in the overall catalytic cycle, oxidation by O2 of the fully reduced oxidase directly followed by re-reduction (R --> O --> R transition), exhibited a bell-shaped pH dependence approaching 4 at pH 7.2. A mechanism for the involvement in the proton pump of the oxidase of H+/e- cooperative coupling at the metal centers (redox Bohr effects) and protonmotive steps of reduction of O2 to H2O is presented.

Anaerobiosis↗
PubMed1996

Structure-based design of an intramolecular proton transfer site in murine carbonic anhydrase V.

Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton. The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme. Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity. The crystal structure of wild-type carbonic anhydrase V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc. We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry. Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer. However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65. This is the first structure-based design of an efficient proton transfer site in an enzyme.

Animals↗
PubMed1996

Kinetics of electron and proton transfer during the reaction of wild type and helix VI mutants of cytochrome bo3 with oxygen.

Site-directed mutagenesis was used to investigate the mechanism of electron and proton transfer in the ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The reaction between the fully reduced form of the enzyme and dioxygen was studied using the flow--flash method. After rapid mixing of CO-bound enzyme with an O2-containing solution, CO was photodissociated, and the subsequent electron- and proton-transfer reactions were measured spectrophotometrically, the latter using a pH-indicator dye. In the wild-type, pure bo3 enzyme, without bound quinones, we observed a single kinetic phase with a rate constant of about 2.4 x 10(4) s-1, associated with formation of the ferry1 oxygen intermediate, followed by proton uptake from solution with a rate constant of about 1.2 x 10(4) s-1. Enzyme in which heme o instead of heme b was incorporated into the low-spin site displayed a slower ferry1 formation with a rate constant of about 3.6 x 10(3) s-1. Upon replacement of the acidic residue glutamate 286 in helix VI of subunit I with a nonprotonatable residue, electron transfer was slightly accelerated, and proton uptake was impaired. Mutations of other residues in the vicinity of E286 also resulted in a dramatic decrease of proton uptake, suggesting that the environment of this residue is important for efficient proton transfer. In the closely related cytochrome aa3 from P. denitrificans, the corresponding residue (E278) has been suggested to be part of a proton-transfer pathway [Iwata, S., Ostermeier, C., Ludwig, B., & Michel, H. (1995) Nature 376, 660-669]. The results are discussed in terms of a model for electron-proton coupling during dioxygen reduction.

Carbon Monoxide↗
PubMed1998

Bacteriorhodopsin's intramolecular proton-release pathway consists of a hydrogen-bonded network.

In its proton-pumping photocycle, bacteriorhodopsin releases a proton to the extracellular surface at pH 7 in the transition from intermediate L to intermediate M. The proton-release group, named XH, was assigned in low-temperature FT-IR studies to a single residue, E204 [Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R. , and Lanyi, J. K. (1995) J. Biol. Chem. 270, 27122-27126]. The time-resolved room-temperature step-scan FT-IR photocycle studies on wild-type and E204Q-, and E204D-mutated bacteriorhodopsin, which we present here, show in contrast that the FT-IR data give no evidence for deprotonation of E204 in the L-to-M transition. Therefore, it is unlikely that E204 represents XH. On the other hand, IR continuum absorbance changes indicate intramolecular proton transfer via an H-bonded network to the surface of the protein. It appears that this H-bonded network is spanned between the Schiff base and the protein surface. The network consists at least partly of internally bound water molecules and is stabilized by E204 and R82. Other not yet identified groups may also contribute. At pH 5, the intramolecular proton transfer to the surface of the protein seems not to be disturbed. The proton seems to be buffered at the surface and later in the photocycle released into the bulk during BR recovery. Intramolecular proton transfer via a complex H-bonded network is proposed to be a general feature of proton transfer in proteins.

Bacteriorhodopsins↗
PubMed2000

Protonation states and pH titration in the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.

Amino Acid Substitution↗
PubMed2004

Electrocatalytic proton reduction by phosphido-bridged diiron carbonyl compounds: distant relations to the H-cluster?

Intermediates formed during reduction of Fe(2)(mu-PPh(2))(2)(CO)(6) (1) in the presence of protons have been identified by spectroelectrochemical, continuous-flow, and interrupted-flow techniques. The mechanism for electrocatalytic proton reduction suggested by these observations yields digital simulation of the voltammetry in close agreement with measurements conducted in THF over a range of acid concentrations. The mechanism for electrocatalytic proton reduction involves initial formation of the dianion, 1(2-), which is doubly protonated prior to further reduction and dihydrogen elimination. The IR spectra of the singly and doubly protonated forms of 1(2-) indicate structures corresponding to [FeH(CO)(3)(mu-PPh(2))(2)Fe(CO)(3)](-) (1H-) and FeH(CO)(3)(mu-PPh(2))(2)FeH(CO)(3) (1H(2)). The thiolato and dithiolato analogues of 1 exhibit electrocatalytic proton reduction associated with the two-electron reduction step, and this implies that the corresponding two-electron reduced doubly protonated species is unstable with respect to dihydrogen elimination. The stability of 1H(2) is most likely to be due to the weak interactions between the iron centers of the flattened [2Fe2P] core. Whereas 1H(2) is stable in the absence of a reducing potential, 1H- rearranges rapidly to a product previously described as [Fe(2)(mu-PPh(2))(mu-CO)(PHPh(2))(CO)(5)](-) (1H-(W)). Another protonation product of 1(2-), previously formulated as [Fe(2)(mu-PPh(2))(2)(mu-CO)H(CO)(5)](-), has been reformulated as [Fe(2)(mu-PPh(2))(mu-CO)(CO)(6)](-) (2) on the basis of a range of spectroscopic measurements. Solution EXAFS measurements of 1, 1(2-), 1H-(W), and 2 are reported, and these yield model-independent Fe-Fe distances of 2.61 (1), 3.58 (1(2-)), 2.58 (1H-(W)), and 2.59 A (2). The presence of an Fe-Fe bond for both 1H-(W) and 2 is a key aspect of the proposed structures, and this strongly supports the deductions based on spectroscopic evidence. The fits of the solution EXAFS to different structural models give statistics in agreement with the proposed structures.

Catalysis↗
PubMed2005

Effect of pressure on proton-coupled electron transfer reactions of seven-coordinate iron complexes in aqueous solutions.

For the first time, the effect of pressure on proton-coupled electron-transfer reactions of two selected seven-coordinate FeIII/II(H2L)(H2O)2 systems [where H2L = 2,6-diacetylpyridine-bis(semicarbazone) and 2,6-diacetylpyridine-bis(semioxamazide), respectively] was examined. The acid-base equilibria of the different Fe(III/II) systems were investigated by spectrophotometric, potentiometric, and electrochemical titrations. On the basis of the obtained species distributions, the pH intervals in which the different protonated forms of the two studied systems exist were defined. In different pH ranges, a different number of protons (from 0 to 3 protons per electron) can be transferred during the redox process, which affects the change in the overall charge on the complexes. For all the different protonation forms of the studied complexes, the change in the redox potentials with pressure was measured and the redox reaction volume was obtained by high-pressure cyclic voltammetry. The results show that in the case of proton-coupled electron transfer, the reaction volume for the neutralization of protons contributes to the overall reaction volume. A linear correlation between Deltaz2 (change in the square of the charge) and the overall reaction volume of the complexes upon reduction, DeltaVcomplex0, was found. The average value of the intrinsic volume change for the selected seven-coordinate iron complexes was estimated from the intercept of the plot of DeltaVcomplex0 versus Deltaz2 to be 9.2 +/- 0.7 cm3 mol(-1). For the combined redox and protonation processes, the data are discussed in terms of linear correlations between Deltaz2 and the redox and neutralization reaction volumes reported in the literature.

Electron Transport↗
PubMed2001

Proton positions in the Mn(2+) binding site of concanavalin A as determined by single-crystal high-field ENDOR spectroscopy.

High-field (95 GHz) pulsed EPR and electron-nuclear double resonance (ENDOR) techniques have been used for the first time to determine coordinates of ligand protons of a high-spin metal center in a protein single crystal. The protein concanavalin A contains a Mn(2+) ion which is coordinated to two water molecules, a histidine residue, and three carboxylates. Single crystals of concanavalin A were grown in H(2)O and in D(2)O to distinguish the exchangeable water protons from the nonexchangeable protons of the imidazole group. Distinct EPR transitions were selected by performing the ENDOR measurements at different magnetic fields within the EPR spectrum. This selection, combined with the large thermal polarization achieved at 4.5 K and a magnetic field of approximately 3.4 T allowed us to assign the ENDOR signals to their respective M(S) manifolds, thus providing the signs of the hyperfine couplings. Rotation patterns were acquired in the ac and ab crystallographic planes. Two distinct crystallographic sites were identified in each plane, and the hyperfine tensors of two of the imidazole protons and the four water protons were determined by simulations of the rotation patterns. All protons have axially symmetric hyperfine tensors and, by applying the point-dipole approximation, the positions of the various protons relative to the Mn(2+) ion were determined. Likewise, the water protons involved in H-bonding to neighboring residues were identified using the published, ultrahigh-resolution X-ray crystallographic coordinates of the protein (Deacon et al. J. Chem. Soc., Faraday Trans. 1997, 93(24), 4305-4312).

Binding Sites↗
PubMed2002

Proton diffusion at phospholipid assemblies.

A new scanning electrochemical microscopy proton feedback method has been developed for investigating lateral proton diffusion at phospholipid assemblies: specifically Langmuir monolayers at the water/air interface. In this approach, a base is electrogenerated by the reduction of a weak acid (producing hydrogen) at a "submarine" ultramicroelectrode (UME) placed in the aqueous subphase of a Langmuir trough close to a monolayer. The electrogenerated base diffuses to and titrates monolayer-bound protons and is converted back to its initial form, so enhancing the current response at the UME. Local deprotonation of the monolayer creates a concentration gradient for lateral proton diffusion. A numerical model has been developed, taking into account the potential-dependent association/dissociation constant of the interfacial acid groups. A comparison is made of monolayers comprising either acidic DL-alpha-phosphatidyl-L-serine, dipalmitoyl (DPPS) or zwitterionic L-alpha-phosphatidylcholine, dipalmitoyl (DPPC) monolayers at a range of surface pressures. It is demonstrated that lateral proton fluxes at DPPS are significant, but the lateral proton diffusion coefficient is lower than in bulk solution. In contrast, lateral proton diffusion cannot be detected at DPPC, suggesting that the acid/base character of the phospholipid is important in determining the magnitude of interfacial proton fluxes.

Diffusion↗
PubMed2002

Theoretical investigation of large kinetic isotope effects for proton-coupled electron transfer in ruthenium polypyridyl complexes.

A theoretical investigation of proton-coupled electron transfer in ruthenium polypyridyl complexes is presented. The three reactions studied are as follows: (1) the comproportionation reaction of [(bpy)(2)(py)Ru(IV)O](2+) and [(bpy)(2)(py)Ru(II)OH(2)](2+) to produce [(bpy)(2)(py)Ru(III)OH](2+); (2) the comproportionation reaction of [(tpy)(bpy)Ru(IV)O](2+) and [(tpy)(bpy)Ru(II)OH(2)](2+) to produce [(tpy)(bpy)Ru(III)OH](2+); and (3) the cross reaction of [(tpy)(bpy)Ru(III)OH](2+) and [(bpy)(2)(py)Ru(II)OH(2)](2+) to produce [(tpy)(bpy)Ru(II)OH(2)](2+) and [(bpy)(2)(py)Ru(III)OH](2+). This investigation is motivated by experimental measurements of rates and kinetic isotope effects for these systems (Binstead, R. A.; Meyer, T. J. J. Am. Chem. Soc. 1987, 109, 3287. Farrer, B. T.; Thorp, H. H. Inorg. Chem. 1999, 38, 2497.). These experiments indicate that the second reaction is nearly one order of magnitude faster than the first reaction, and the third reaction is in the intermediate regime. The experimentally measured kinetic isotope effects for these three reactions are 16.1, 11.4, and 5.8, respectively. The theoretical calculations elucidate the physical basis for the experimentally observed trends in rates and kinetic isotope effects, as well as for the unusually high magnitude of the kinetic isotope effects. In this empirical model, the proton donor-acceptor distance is predicted to be largest for the first reaction and smallest for the third reaction. This prediction is consistent with the degree of steric crowding near the oxygen proton acceptor for the three reactions. The second reaction is faster than the first reaction since a smaller proton donor-acceptor distance leads to a larger overlap between the reactant and product proton vibrational wave functions. The intermediate rate of the third reaction is determined by a balance among several competing factors. The observed trend in the kinetic isotope effects arises from the higher ratio of the hydrogen to deuterium vibrational wave function overlap for larger proton donor-acceptor distances. Thus, the kinetic isotope effect increases for larger proton donor-acceptor distances. The unusually high magnitude of the kinetic isotope effects is due in part to the close proximity of the proton transfer interface to the electron donor and acceptor. This proximity results in strong electrostatic interactions that lead to a relatively small overlap between the reactant and product vibrational wave functions.

Deuterium↗
PubMed2003

Kinetic isotope effects for concerted multiple proton transfer: a direct dynamics study of an active-site model of carbonic anhydrase II.

The rate constant of the reaction catalyzed by the enzyme carbonic anhydrase II, which removes carbon dioxide from body fluids, is calculated for a model of the active site. The rate-determining step is proton transfer from a zinc-bound water molecule to a histidine residue via a bridge of two or more water molecules. The structure of the active site is known from X-ray studies except for the number and location of the water molecules. Model calculations are reported for a system of 58 atoms including a four-coordinated zinc ion connected to a methylimidazole molecule by a chain of two waters, constrained to reproduce the size of the active site. The structure and vibrational force field are calculated by an approximate density functional treatment of the proton-transfer step at the Self-Consistent-Charge Density Functional Tight Binding (SCC-DFTB) level. A single transition state is found indicating concerted triple proton transfer. Direct-dynamics calculations for proton and deuteron transfer and combinations thereof, based on the Approximate Instanton Method and on Variational Transition State Theory with Tunneling Corrections, are in fair agreement and yield rates that are considerably higher and kinetic isotope effects (KIEs) that are somewhat higher than experiment. Classical rate constants obtained from Transition State Theory are smaller than the quantum values but the corresponding KIEs are five times larger. For multiple proton transfer along water bridges classical KIEs are shown to be generally larger than quantum KIEs, which invalidates the standard method to distinguish tunneling and over-barrier transfer. In the present case, a three-way comparison of classical and quantum results with the observed data is necessary to conclude that proton transfer along the bridge proceeds by tunneling. The results suggest that the two-water bridge is present in low concentrations but makes a substantial contribution to proton transport because of its high efficiency. Bridging structures containing more water molecules may have lower energies but are expected to be less efficient. The observed exponential dependence of the KIEs on the deuterium concentration in H(2)O/D(2)O mixtures implies concerted transfer and thus rules out substantial contributions from structures that lead to stepwise transfer via solvated hydronium ions, which presumably dominate proton transfer in less efficient carbonic anhydrase isozymes.

Binding Sites↗
PubMed2004

Dynamics of proton transfer in bacteriorhodopsin.

Proton transfer in bacteriorhodopsin from the cytoplasm to the extracellular side is initiated from protonated asp96 in the cytoplasmic region toward the deprotonated Schiff base. This occurs in the transition from the photocycle late M state to the N state. To investigate this proton-transfer process, a quantum mechanics/molecular mechanics (QM/MM) model is constructed from the bacteriorhodopsin E204Q mutant crystal structure. Three residues, asp96, asp85, and thr89, as well as most of the retinal chromophore and the Schiff base link of lys216 are treated quantum mechanically and connected to the remaining classical protein through linker atom hydrogens. Structural transformation in the M state results in the formation of a water channel between the Schiff base and asp96. Since a part of this channel is lined with hydrophobic residues, there has been a question on the mechanism of proton transfer in a hydrophobic channel. Ab initio dynamics using the CHARMM/GAMESS methodology is used to simulate the transfer of the proton through a partially hydrophobic channel. Once sufficient water molecules are added to the channel to allow the formation of a single chain of waters from asp96 to the Schiff base, the transfer occurs as a fast (less than a picosecond) concerted event irrespective of the protonation state of asp85. Dynamic transfer of the proton from asp96 to the nearest water initiates the organization of a strongly bonded water chain conducive to the transfer of the proton to the Schiff base nitrogen.

Bacteriorhodopsins↗
PubMed2004

Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that 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 remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.

Animals↗
PubMed2006

Proton transfer-induced conformational changes and melting in designed peptides in the gas phase.

The conformations of protonated RA15K, RA20K and RA15H (R = arginine, A = alanine, K = lysine, and H = histidine) have been examined in the gas phase as a function of temperature. These peptides were designed so that intramolecular proton transfer will trigger conformational changes between a helix (proton sequestered at the C-terminus) and globule (proton sequestered at the N-terminus). Kinetically controlled structural transitions occur below 400 K (from helix to globule for RA15H, and from globule to helix for RA15K and RA20K). As the temperature is raised, the compact globule found at room temperature expands, accesses more configurations, and becomes entropically favored. At around 500 K, the RA15K and RA20K helices undergo a melting transition. The transition is broad, as expected for a phase transition in a finite system, and becomes narrower as the peptide size increases. In the helical conformation, the two basic residues are well separated; as a result, the proton transfer necessary to drive the melting transition probably involves a mobile proton. For doubly protonated RA15K, a dumbbell-like conformation (resulting from repulsion between the two protonated basic residues) is found at high temperature.

Gases↗
PubMed2006

Control of stereoselective protonation of enols1,2.

A decalyl framework with a siloxy enolic moiety and proximate proton transferring groups was synthesized. On enolate generation with fluoride two competitive reaction modes were possible: (a) intermolecular protonation, and (b) intramolecular proton transfer by the proximate group. Control of the protonation stereochemistry proved possible by varying the proximate group and by changing the acidity of the medium. With the groups -CH2OH, -CH=O, and -CH2OCH2OCH3 as the proximate groups, only intermolecular proton transfer was observed with no dependence on acidity. In contrast, with -COO- and COOH, only intramolecular protonation resulted but again with no dependence on acidity of the medium. In contrast, with -CH2NH2 as the proximate group, intramolecular proton transfer predominated with a dependence on the effective pH of the medium. A kinetic analysis provided a linear-log relationship of the ratio of the two stereoisomers with the medium acidity. The analysis revealed that two acetic acid molecules are involved in providing the proton to the enolate moiety. A theoretical analysis was developed paralleling the experimental results. In the ketonization transition state, the hybridization was shown to be close to sp2 hybridized at the alpha-enolate carbon.

Acetic Acid↗
PubMed2006

Carbon protonation of 2,4,6-triaminopyrimidines: synthesis, NMR studies, and theoretical calculations.

Several C5-substituted 2,4,6-triaminopyrimidine derivatives and their HBF4 salts were synthesized to study the carbon protonation of the pyrimidine ring. NMR investigations in DMSO-d6 prove experimentally that, in addition to the usual protonation at N1, the compounds can be protonated at C5 as well. We present several new stable cationic sigma-complexes in the pyrimidine series, where C5 protonation predominates over N1 protonation. Quantum chemical calculations using the B3LYP/cc-pVDZ method were utilized in the gas phase and also in DMSO solvent with the polarized continuum model (PCM) method to rationalize the observed protonation behavior. Results of the calculations accord with the experimental observations and prove that combined steric and electronic effects are responsible for the observed C5 protonation and for sigma-complex stability. We demonstrate that C5 protonation is a general feature of the 2,4,6-triaminopyrimidine system.

Carbon↗
PubMed2003

Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation.

The Na+/H+ exchanger is an integral membrane protein found in the plasma membrane of eukaryotic and prokaryotic cells. In eukaryotes it functions to exchange one proton for a sodium ion. In mammals it removes intracellular protons while in plants and fungal cells the plasma membrane form removes intracellular sodium in exchange for extracellular protons. In this study we used the Na+/H+ exchanger of Schizosaccharomyces pombe (Sod2) as a model system to study amino acids critical for activity of the protein. Twelve mutant forms of the Na+/H+ exchanger were examined for their ability to translocate protons as assessed by a Cytosensor microphysiometer. Mutation of the amino acid Histidine 367 resulted in defective proton translocation. The acidic residues Asp145, Asp178, Asp266 and Asp267 were important in the proton translocation activity of the Na+/H+ exchanger. Mutation of amino acids His98, His233 and Asp241 did not significantly impair proton translocation by the Na+/H+ exchanger. These results confirm that polar amino acids are important in proton flux activity of Na+/H+ exchangers.

Amino Acids↗
PubMedDate not supplied

Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel.

Protons participate in most biologically important reactions, as substrates, products, cofactors and modulators, and proton transport is an essential step in energy transduction. The dynamics of protonation reactions have been studied extensively in solution and in model systems involving lipid-water interfaces, but have never been resolved at the timescale of the elementary molecular event. Here we show that, under appropriate conditions, binding and unbinding reactions of single protons and deuterium ions to a single site on the L-type calcium channel can be resolved and the protonation and deprotonation rates quantified. The protonation rate constant considerably exceeds previous estimates obtained in simpler systems. The functional consequences of channel protonation is a threefold reduction of the channel conductance, independent of the applied voltage. The data are consistent with the presence of a single protonatable group with pK in the physiological pH range, close to the external mouth of the channel. The two conductance levels of the open channel might be explained by greatly differing local potentials associated with the protonated and deprotonated state of the group.

Animals↗