Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. II. Interaction with a bulk proton.

The reaction mechanism and the dynamic aspects of protonation of a defined moiety located inside an aqueous cavity in a protein were monitored by time resolved spectroscopy using the pyranine apomyoglobin complex as a model (Shimoni, Tsfadia, Nachliel, and Gutman, 1993, Biophys. J. 64:472-479). The reaction was synchronized by a short laser pulse and the reprotonation of the ground state pyranine anion (phi O-) was monitored, in the microsecond time scale, by its transient absorption at 457 nm. The observed signal was reconstructed by a numeric solution of nonlinear, coupled differential equations which account for the direct reaction of phi O- with bulk proton and by proton transfer from the nearby amino acids: His 64, Asp 44, Asp 60, and Glu 59. A unique combination of rate constant was obtained which quantitates the contribution of each pathway to the overall relaxation process. In the first phase of the dynamics phi O- abstracts a proton from the nearby protonated histidine. The bulk proton interacts preferentially with the cluster of three carboxylates and immediately shuttled to the deprotonated histidine. The high proximity of the reactive groups and the strong electrostatic forces operating inside the heme binding cavity render the rate of proton transfer in the site ultrafast.     

Quantitation of physical-chemical properties of the aqueous phase inside the phoE ionic channel.

The anion-specific channel of the phoE porine is a miniature body of water surrounded by peptide walls. The physical and chemical properties of the water in such a microscopic space were measured by monitoring the dynamics of a well-studied reaction--the protolytic dissociation of a strong acid. To attain this purpose, we allowed pyranine (8-hydroxypyrene-1,3,6-trisulfonate) to bind to the anion-specific channel. The dye is bound, with a 1:1 stoichiometry, with a delta G = -9.5 kcal/mol. Photoexcitation of the dye, to its first electronic singlet state (phi OH*), renders it very acidic and the hydroxyl proton dissociates to H+ and excited anion (phi O*-). We employed single photon-counting time-resolved fluorimetry, to monitor the reversible dissociation of pyranine as it proceeds within the channel and reconstructed the observed signal by a numerical integration of the differential diffusion equation pertinent for a proton within the channel. The most characteristic feature of the water-filled channel, is the intensified electrostatic interactions attained by the low dielectric constant of the diffusion space, epsilon eff = 24. For this reason, the electric field of a few positive charges is sufficient to ensure that an anion entering the channel will be effectively sucked in. The interaction of the water molecules with the peptide structure forming the channel affects the physical properties of the water. Their capacity to conduct proton, quantitated by the protons diffusion coefficient (4.5.10(-5) cm2/s), is reduced by 50% with respect to that of bulk water. The activity of the water in the channel is reduced to alpha H2O = 0.966. These observation are in accord with our previous studies of water in small defined cavities in proteins.     

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles

Summary The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3–10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986.Methods Enzymol. 127:400–416).In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986.Methods Enzymol. 127:522–538).In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state.Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage.The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer.The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.     

Dynamics of the proton transfer reaction on the cytoplasmic surface of bacteriorhodopsin

The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to approximately 60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.     

Gaugement of the inner space of the apomyoglobin's heme binding site by a single free diffusing proton. I. Proton in the cavity.

Time resolved fluorimetry was employed to monitor the geminate recombination between proton and excited pyranine anion locked, together with less than 30 water molecules, inside the heme binding site of Apomyoglobin (sperm whale). The results were analyzed by a numerical reconstruction of the differential rate equation for time-dependent diffusion controlled reaction with radiating boundaries using N. Agmon's procedure (Huppert, Pines, and Agmon, 1990, J. Opt. Soc. Am. B., 7:1541-1550). The analysis of the curve provided the effective dielectric constant of the proton permeable space in the cavity and the diffusion coefficient of the proton. The electrostatic potential within the cavity was investigated by the equations given by Gilson et al. (1985, J. Mol. Biol., 183:503-516). According to this analysis the dielectric constant of the protein surrounding the site is epsilon prot < or = 6.5. The diffusion coefficient of the proton in the heme binding site of Apomyoglobin-pyranine complex is D = 4 x 10(-5) cm2/s. This value is approximately 50% of the diffusion coefficient of proton in water. The lower value indicates enhanced ordering of water in the cavity, a finding which is corroborated by a large negative enthropy of binding delta S0 = -46.6 cal.mole-1 deg-1. The capacity of a small cavity in a protein to retain a proton had been investigated through the mathematical reconstruction of the dynamics. It has been demonstrated that Coulombic attraction, as large as delta psi of energy coupling membrane, is insufficient to delay a free proton for a time frame comparable to the turnover time of protogenic sites.     

Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H(+)-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.     

Proton binding to biological membranes

Biological membranes contain proton-binding moieties. A laser-induced proton pulse was used to characterize the proton-binding properties of bacterioopsin-containing membranes and of sarcoplasmic reticulum. Different protonation and deprotonation processes occurred. The liberation of protons from pyranine dye and the protonation of the membranes were independent of temperature; the reprotonation of pyranine and proton release from the membranes were temperature dependent. In the cases of membrane-free and membrane-containing systems, the activation enthalpies and entropies were calculated from the decay rates. The activation enthalpy of 16 kJ/mol for reprotonation of pyranine in membrane-free solution is characteristic for a diffusion-controlled process. The value for the membrane-containing systems was nearly double, suggesting that the buffering moieties of the membrane surfaces strongly bind the protons, raising the activation enthalpies. This is possibly an effect of the Coulomb cages formed from closely located proton acceptor sites. The activation entropies were positive in all cases.     

Pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) as a probe of internal aqueous hydrogen ion concentration in phospholipid vesicles

The fluorescence intensity (at 510 nm) of the hydrophilic pyrene analogue 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine) is strongly dependent upon the degree of ionization of the 8-hydroxyl group (pKa = 7.2) and hence upon the medium pH, over the range pH 6--10. Because of its polyanionic character, pyranine does not bind significantly to phospholipid vesicles having a net anionic surface charge. As a result, it is possible to form vesicles in the presence of pyranine which, after removal of external probe by gel filtration, contain pyranine entrapped within the internal aqueous compartment. Once entrapped, pyranine does not readily leak out of the vesicles. Because the fluorescence properties of entrapped pyranine resemble closely the properties of bulk pyranine solution with respect to pH sensitivity, pyranine can be used as a reliable reporter of aqueous pH changes within anionic vesicles. When HCl is rapidly added to a suspension of unilamellar soybean phospholipid (asolectin) vesicles preincubated at alkaline pH, a biphasic decrease in the pH of the vesicle inner aqueous compartment is observed. An initial, very rapid and electrically uncompensated H+ influx (t 1/2 less than 1 s) results in the generation of a transmembrane electric potential opposing further H+ influx. This leads to the development of a much slower (t 1/2 approximately equal to 5 min), valinomycin-sensitive, proton--counterion exchange which continues until the proton concentration gradient is eliminated. Similar results were obtained in asolectin vesicles prepared by detergent dilution, in sonicated egg phosphatidylcholine vesicles, and in multilamellar asolectin liposomes. The rather high permeability of soybean lipid membranes to H+ is surprising in view of the widespread use of these lipids for the reconstitution of membrane proteins which are thought to generate or utilize H+ ion gradients in energy transduction reactions.     

Subsecond proton-hole propagation in bacteriorhodopsin

The dynamics of proton transfer between the surface of purple membrane and the aqueous bulk have recently been investigated by the Laser Induced Proton Pulse Method. Following a Delta-function release of protons to the bulk, the system was seen to regain its state of equilibrium within a few hundreds of microseconds. These measurements set the time frame for the relaxation of any state of acid-base disequilibrium between the bacteriorhodopsin's surface and the bulk. It was also deduced that the released protons react with the various proton binding within less than 10 micro s. In the present study, we monitored the photocycle and the proton-cycle of photo-excited bacteriorhodopsin, in the absence of added buffer, and calculated the proton balance between the Schiff base and the bulk phase in a time-resolved mode. It was noticed that the late phase of the M decay (beyond 1 ms) is characterized by a slow (subsecond) relaxation of disequilibrium, where the Schiff base is already reprotonated but the pyranine still retains protons. Thus, it appears that the protonation of D96 is a slow rate-limiting process that generates a "proton hole" in the cytoplasmic section of the protein. The velocity of the hole propagation is modulated by the ionic strength of the solution and by selective replacements of charged residues on the interhelical loops of the protein, at domains that seems to be remote from the intraprotein proton conduction trajectory.     

Dynamic studies of proton diffusion in mesoscopic heterogeneous matrix: II. The interbilayer space between phospholipid membranes

The thin water layer, as found in chloroplast or mitochondria, is confined between low dielectric amphypathic surfaces a few nm apart.

The physical properties of this mesoscopic space, and how its dimensions affect the rate of chemical reactions proceeding in it, is the subject for this study.

The method selected for this purpose is time resolved fluorometry which can monitor the reversible dissociation of a proton from excited molecule of pyranine (8 hydroxy pyrene 1,3,6 tri sulfonate) trapped in thin water layers of a multilamellar vesicle made of neutral or slightly charged phospholipids.

The results were analyzed by a computer program of N. Agmon (Pines, E., D. Huppert, and N. Agmon. 1988. J. Am. Chem. Soc. 88:5620-5630) that simulates the diffusion of a proton, subjected to electrostatic attraction, in a thin water layer enclosed between low affinity, proton binding surfaces. The analysis determines the diffusion coefficient of the proton, the effective dielectric constant of the water and the water accessibility of the phosphomoieties of the lipids.

These parameters were measured for various lipids [egg-phosphatidylcholine (egg PC), dipalmitoyl phosphatidylcholine (DPPC), cholesterol + DPPC (1:1) and egg PC plus phosphatidyl serine (9:1)] and under varying osmotic pressure which reduces the width of the water layer down to ~10 ~ across.

We found that: (a) The effective dielectric constant of the aqueous layer, depending on the lipid composition, is ~40. (b) The diffusion coefficient of the proton in the thin layer (30-10 ~ across) is that measured in bulk water D = 9.3 10^-5 cm²/s, indicating that the water retains its normal liquid state even on contact with the membrane. (c) The reactivity of the phosphomoiety, quantitated by rate of its reaction with proton, diminishes under lateral pressure which reduces the surface area per lipid.

We find no evidence for abnormal dynamics of proton transfer at the lipid water interface which, by any mechanism, accelerates its diffusion.

     

Viscosity of the internal aqueous phase of unilamellar phospholipid vesicles

The viscosity of the internal aqueous phase of unilamellar vesicles comprised of purified soybean phospholipids (asolectin) was measured using the fluorescence polarization of the entrapped hydrophilic probe 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine). At 20 °C the rotational relaxation time (?) for pyranine in bulk solution was 0.55 ns as compared to a rotational time of 1.8 ns for pyranine within the internal aqueous compartment. Similar large increases in ? for internal pyranine were noted over the temperature range 5–35 °C suggesting that in these small vesicles the internal water is more viscous than bulk water.     

Proton transfer dynamics on the surface of the late M state of bacteriorhodopsin

The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.     

The effect of solvent viscosity on the rate-determining step of fatty acid synthetase

The overall activity of an animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, eta, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to eta-P, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p less than 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5-fold increase of solvent viscosity with sucrose. These results suggested that the rate-determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate-determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to eta, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity-dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by beta-ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate-determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics,mechanism, and regulation

Cotyledons of Ricinus communis take up externally supplied sucrose at a rate of up to 150 mol/h/g fresh weight, which is very high when compared with other sugar transport systems of higher plants. The uptake of sucrose is catalysed with a K _m of 25 mmol l^–1; at high sucrose concentrations a linear (diffusion) component becomes obvious. Other mono-, di-, or trisaccharides do not compete for sucrose uptake. Sucrose is accumulated by the cotyledons up to 100-fold, whereby most of the transported, externally supplied sucrose mixes with sucrose present in the tissue. At low sucrose concentrations, however; a small unexchangeable internal pool of sucrose becomes evident. Poisons of energy metabolism such as FCCP inhibit uptake and accumulation of sucrose. The transport of sucrose induces an increase of respiration, from which an energy requirement of 1.4 ATP/sucrose taken up can be calculated. Sucrose is taken up together with protons at an apparent stoichiometry of 0.3 protons/sucrose. Other sugars do not cause proton uptake. The K _m for sucrose induced proton uptake is 5 mmol l^–1; the discrepancy to the K _m for sucrose uptake as well as the low proton: sucrose stoichiometry might possibly be caused by a large contribution of diffusion barriers. The estimated proton-motive potential difference would by sufficient to explain an electrogenic sucrose accumulation. The rate of uptake of sucrose is subject to feedback inhibition by internal sucrose. It is also regulated during growth of the seedlings since it develops rapidly during the first days of germination and declines again after the 4th day of germination, though no substantial increase of passive permeability resistance was observed.Abbreviations DMO dimethyloxazolidinedione - FCCP trifluoromethoxy (carbonyl-cyanide) phenylhydrazon - fr. wt. fresh weight     

On the mechanism of nitrobenzene liquid membrane oscillators containing hexadecyltrimethylammonium bromide

The oscillatory behaviour of a liquid membrane oscillator was investigated in order to contribute to the oscillation mechanism at the molecular level. The chosen system involved nitrobenzene as liquid membrane containing a constant amount of picric acid. The aqueous donor phase contained the cationic surfactant, hexadecyltrimethylammonium bromide, and ethanol. The aqueous acceptor phase was made up by sucrose solution. It was established that the oscillations take place exclusively at the aqueous acceptor phase/membrane interface. Three stages mechanism of the oscillation (I--induction period, II--first peak formation, III--creation of the first peak) together with appropriate processes were proposed. The molecular events provoking the oscillations of electric potential difference between the two aqueous phases concern: 1) the diffusion of hexadecyltrimethylammonium bromide and ion pairs formed by cation of the surfactant and the picrate anion to the vicinity of the membrane/acceptor phase interface, 2) sudden adsorption of these ion-pairs at this interface in non-catalytic and autocatalytic steps, 3) desorption of ion pairs from the a/m interface to the acceptor phase. It is shown by numerical simulations that the proposed mechanism may account for the observed oscillations and for the species distribution throughout the system as found experimentally.     

Dynamic aspect of kinetics of reaction catalyzed by lactate dehydrogenase

The influence of solvent viscosity on the kinetic parameters of the pyruvate reduction reaction catalyzed by lactate dehydrogenase has been investigated. The viscosity was adjusted by sucrose and glycerol solutions at concentrations from 0 to 44% and from 0 to 63%, respectively. The reaction rate decreased abruptly with an increase in viscosity. The study of different reaction stages (enzyme-substrate complex formation, catalysis, inhibitory complex decomposition, competitive inhibition by chlorine ions) revealed that the catalysis (and the related conformational changes) is the only stage (of the above mentioned) that depends markedly on the solvent viscosity. The reaction is insensitive to the changes in the dielectric properties of the solution induced by the addition of alcohols and dioxane. The observed power dependence of the rate constant on viscosity is explained in terms of Kramer's theory which considers the proton transition through the activation barrier to be a diffusion in the field of random forces. The influence of solvent viscosity on enzymic kinetics indicates a direct relation between solvent dynamics and relevant protein conformational movements.     

Proton transfer dynamics in the nonhomogeneous electric field of a protein.

By adsorption of pyranine (8 hydroxypyrene 1, 3, 6 trisulfonate) to lysozyme we create on the positively charged protein a fluorophoric site with a total charge of -3. Photo dissociation of the dye's hydroxyl proton changes its absorption and fluorescence spectrum, permitting a continuous monitoring of the reprotonation dynamics. Absorbance measurements in the microsecond time scale monitor how the bulk protons penetrate the Coulomb cage of the bound dye. Time-resolved fluorescence monitors how the proton is escaping out of the Coulomb cage of the bound dye. These probe reactions were studied with a series of dye-enzyme complexes where the number of free carboxylate was reduced by amidation, increasing the total charge of the complex from +5 to +12.6. The time-resolved measurements demonstrate the complexity of the electric field in the immediate vicinity of the dye. It is consistent with a negative potential wall (of the anion) surrounded by a positive potential wall of proteinaceous moieties.     

A picosecond time-resolved study on prototropic reactions of electronically excited 1,5- and 1,8-diaminonaphthalenes in aqueous solution.

The proton transfer to solvent in the excited state of protonated diaminonaphthalenes, 1,5-diaminonaphthalene (1,5-DAN) and 1,8-diaminonaphthalene (1,8-DAN), in aqueous solution, has been investigated by picosecond time-resolved fluorescence measurements. The deprotonation rate constants of the dications of 1,8-DAN and 1,5-DAN in the excited state to produce the corresponding monocations are determined to be 1.3 x 10(10) and 5.6 x 10(9) s(-1), respectively, from dynamic analyses of their fluorescence time profiles. The much larger proton-dissociation rates compared with that of 1-aminonaphthalene (0.6 x 10(9) s(-1)) can be attributed to an electron-withdrawing effect due to the ammonium group at the 5- or 8-position in the naphthalene ring. The remarkably large proton-dissociation rate in 1,8-DAN can be ascribed to its larger reaction exergonicity which results from the electrostatic repulsion between the two ammonium groups in the reactant (the dication state) and the stabilization of the monocation state due to hydrogen bonding interactions between the NH3+ and NH2 moieties. The difference in their acidities in the excited state is discussed in terms of the reaction free energy and the proton affinities are evaluated from ab initio MO calculations.     

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression—an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression—through fast promoting vibrations, or other means—could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.     

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)