Reaction within the coulomb-cage; science in retrospect.

The Coulomb-cage is defined as the space where the electrostatic interaction between two bodies is more intensive than the thermal energy (kBT). For small molecule, the Coulomb-cage is a small sphere, extending only few water molecules towards the bulk and its radius is sensitive to the ionic strength of the solution. For charged proteins or membranal structures, the Coulomb-cage can engulf large fraction of the surface and provides a preferred pathway for ion propagation along the surface. Similarly, electrostatic potential at the inner space of a channel can form preferential trajectories passage for ions. The dynamics of ions inside the Coulomb-cage of ions was formulated by the studies of proton-anion recombination of excited photoacids. In the present article, we recount the study of intra- Coulomb-cage reaction taking place on the surface of macro-molecular bodies like micelles, membranes, proteins and intra-protein cavities. The study progressed stepwise, tracing the dynamics of a proton ejected from a photo-acid molecule located at defined sites (on membrane, inter-membrane space, active site of enzyme, inside Large Pore Channels etc.). Accumulation of experimental observations encouraged us to study of the reaction mechanism by molecular dynamics simulations of ions within the Coulomb-cage of proteins surface or inside large pores. The intra-Coulomb-cage proton transfer events follows closely the fine structure of the electrostatic field inside the cage and reflects the shape of nearby dielectric boundaries, the temporal ordering of the solvent molecules and the structural fluctuations of the charged side chains. The article sums some 40 years of research, which in retrospect clarifies the intra-Coulomb-cage reaction mechanism.

The dynamics of proton transfer between adjacent sites.

The mechanism of proton transfer at the interface is the most prevalent reaction in the biosphere, yet its modeling at atomic level is still technically impossible. The difficulties emerge from the quantum mechanical nature of the proton, the modulation of the local electrostatic potential by the protein-water dielectric boundary and the formation of covalent bonds with proton binding sites whenever encounters take place. To circumvent some of these difficulties, and to identify the effect of the local electrostatic field, we present molecular dynamics simulations, where Na+ and Cl- ions diffuse at the surface of a small model protein, the S6 of the bacterial ribosome. The analysis reveals the presence of a detained state, where an ion is located for a relatively long period within the immediate environment of certain attractor residues. In the detained state the ion retains its ability to diffuse, yet the local field deters it from leaving to the bulk. When an ion is detained inside a Coulomb cage, it has a high probability to be transferred between nearby attractors, thus forming a mechanism similar to that responsible for the proton collecting antenna present on proton proteins.

Determination of a unique solution to parallel proton transfer reactions using the genetic algorithm.

Kinetic analysis of the dynamics as measured in multiequilibria systems is readily attained by curve-fitting methodologies, a treatment that can accurately retrace the shape of the measured signal. Still, these reconstructions are not related to the detailed mechanism of the process. In this study we subjected multiple proton transfer reactions to rigorous kinetic analysis, which consists of solving a set of coupled-nonlinear differential rate equations. The manual analysis of such systems can be biased by the operator; thus the analysis calls for impartial corroboration. What is more, there is no assurance that such a complex system has a unique solution. In this study, we used the Genetic Algorithm to investigate whether the solution of the system will converge into a single global minimum in the multidimensional parameter space. The experimental system consisted of proton transfer between four proton-binding sites with seven independent adjustable parameters. The results of the search indicate that the solution is unique and all adjustable parameters converge into a single minimum in the multidimensional parameter space, thus corroborating the accuracy of the manual analysis.

Proton-collecting properties of bovine heart cytochrome C oxidase: kinetic and electrostatic analysis.

Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.

Probing of the substrate binding domain of lactose permease by a proton pulse.

The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.

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.

Time-resolved study of the inner space of lactose permease.

Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1). The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.

Theoretical calculations of the permeability of monensin-cation complexes in model bio-membranes.

Monensin is one of the best-characterized ionophores; it functions in the electroneutral exchange of cations between the extracellular and cytoplasmic sides of cell membranes. The X-ray crystal structures of monensin in free acid form and in complex with Na(+), K(+) and Ag(+) are known and we have recently measured the diffusion rates of monensin in free acid form (Mo-H) and in complex with Na(+) (Mo-Na) and with K(+) (Mo-K) using laser pulse techniques. The results have shown that Mo-H diffuses across the membrane one order of magnitude faster than Mo-Na and two orders of magnitude faster than Mo-K. Here, we report calculations of the translocation free energy of these complexes across the membrane along the most favorable path, i.e. the lowest free energy path. The calculations show that the most favorable orientation of monensin is with its hydrophobic furanyl and pyranyl moieties in the hydrocarbon region of the membrane and the carboxyl group and the cation at the water-membrane interface. Further, the calculations show that Mo-H is likely to be inserted deeper than Mo-Na into the bilayer, and that the free energy barrier for transfer of Mo-H across the membrane is approximately 1 kcal/mol lower than for Mo-Na, in good agreement with our measurements. Our results show that the Mo-K complex is unlikely to diffuse across lipid bilayers in its X-ray crystal structure, in contrast to the Mo-H and Mo-Na complexes. Apparently, when diffusing across the membrane, the Mo-K complex assumes a different conformation and/or thinning defects in the bilayer lower significantly the free energy barrier for the process. The suitability of the model for treating the membrane association of small molecules is discussed in view of the successes and failures observed for the monensin system.

Biophysical aspects of intra-protein proton transfer.

The passage of proton trough proteins is common to all membranal energy conserving enzymes. While the routes differ among the various proteins, the mechanism of proton propagation is based on the same chemical-physical principles. The proton progresses through a sequence of dissociation association steps where the protein and water molecules function as a solvent that lowers the energy penalty associated with the generation of ions in the protein. The propagation of the proton in the protein is a random walk, between the temporary proton binding sites that make the conducting path, that is biased by the intra-protein electrostatic potential. Kinetic measurements of proton transfer reactions, in the sub-ns up to micros time frame, allow to monitor the dynamics of the partial reactions of an overall proton transfer through a protein.

The proton collecting function of the inner surface of cytochrome c oxidase from Rhodobacter sphaeroides.

The experiments presented in this study address the problem of how the cytoplasmic surface (proton-input side) of cytochrome c oxidase interacts with protons in the bulk. For this purpose, the cytoplasmic surface of the enzyme was labeled with a fluorescein (Flu) molecule covalently bound to Cys223 of subunit III. Using the Flu as a proton-sensitive marker on the surface and phiOH as a soluble excited-state proton emitter, the dynamics of the acid-base equilibration between the surface and the bulk was measured in the time-resolved domain. The results were analyzed by using a rigorous kinetic analysis that is based on numeric integration of coupled nonliner differential rate equations in which the rate constants are used as adjustable parameters. The analysis of 11 independent measurements, carried out under various initial conditions, indicated that the protonation of the Flu proceeds through multiple pathways involving diffusion-controlled reactions and proton exchange among surface groups. The surface of the protein carries an efficient system made of carboxylate and histidine moieties that are sufficiently close to each other as to form a proton-collecting antenna. It is the passage of protons among these sites that endows cytochrome c oxidase with the capacity to pick up protons from the buffered cytoplasmic matrix within a time frame compatible with the physiological turnover of the enzyme.

Mechanism of proton entry into the cytoplasmic section of the proton-conducting channel of bacteriorhodopsin.

Bacteriorhodopsin is the light-driven proton-pumping protein of Halobacterium salinarum that extracts protons from the well-buffered cytoplasmic space within the time limits set by the photocycle turnover. The specific mechanism of the proton uptake by the cytoplasmic surface of the protein was investigated in this study by the laser-induced proton pulse technique. The purple membrane preparations were labeled by fluorescein at two residues (36 or 38) of the cytoplasmic surface of the protein, sites that are close to the orifice of the proton-conducting channel. The membranes were pulsed by protons discharged from photoexcited pyranine [Nachliel, E., Gutman, M., Kiryati, S., and Dencher, N.A. (1996) Proc. Nat Acad. Sci. U.S.A. 93, 10747-10752). The reaction of the discharged protons with the pyranine anion and the fluorescein was measured with sub-microsecond resolution. The experimental signals were reconstructed through numeric integration of differential rate equations which quantitated the rates of all proton transfer reactions between all reactants present in the system. The interaction of protons with the orifice of the cytoplasmic channel is enhanced by the exposed carboxylates of the protein. A cluster of three carboxylates acts as a strong proton attractor site while one carboxylate, identified as D36, acts as a mediator that delivers the proton to the channel. The combination of these reactions render the surface of the protein with properties of a proton-collecting antenna. The size of the collecting area is less than that of the protein's surface.

Time-resolved dynamics of proton transfer in proteinous systems.

The dynamics of proton dissociation from an acidic moiety and its subsequent dispersion in the bulk is regulated by the physical chemical properties of the solvent. The solvent has to provide a potential well to accommodate the discharged proton, screen it from the negative charge of the conjugated base, and provide an efficient mode for the diffusion of the proton to the bulk. On measuring the dynamics of proton dissociation in the time-resolved domain, the kinetic analysis of the reaction can quantitate the properties of the immediate environment. In this review we implement the kinetic analysis for evaluating the properties of small cavities in proteins and the diffusion of protons within narrow channels. On the basis of this analysis,we discuss how the clustering of proton-binding sites on a surface can endow the surface with enhanced capacity to attract protons and to funnel them toward a specific site.

The mechanism of monensin-mediated cation exchange based on real time measurements.

Monensin is an ionophore that supports an electroneutral ion exchange across the lipid bilayer. Because of this, under steady-state conditions, no electric signals accompany its reactions. Using the Laser Induced Proton Pulse as a synchronizing event we selectively acidify one face of a black lipid membrane impregnated by monensin. The short perturbation temporarily upsets the acid-base equilibrium on one face of the membrane, causing a transient cycle of ion exchange. Under such conditions the molecular events could be discerned as a transient electric polarization of the membrane lasting approx. 200 microseconds. The proton-driven chemical reactions that lead to the electric signals had been reconstructed by numeric integration of differential rate equations which constitute a maximalistic description of the multi equilibria nature of the system (Gutman, M. and Nachliel, E. (1989) Electrochim. Acta 34, 1801-1806). The analysis of the reactions reveals that the ionic selectivity of the monensin (H+ > Na+ > K+) is due to more than one term. Besides the well established different affinity for the various cations, the selectivity is also derived from a large difference in the rates of cross membranal diffusivities (MoH > MoNa > MoK), which have never been detected before. (v) Quantitative analysis of the membrane's crossing rates of the three neutral complexes reveals a major role of the membranal dipolar field in regulating ion transport. The diffusion of MoH, which has no dipole moment, is hindered only by the viscose drag. On the other hand, the dipolar complexes (MoNa and MoK) are delayed by dipole-dipole interaction with the membrane. (vi) Comparison of the calculated dipoles with those estimated for the crystalline conformation of the [MoNa(H2O)2] and [MoK(H2O)2] complexes reveals that the MoNa may exist in the membrane at its crystal configuration, while the MoK definitely attains a structure having a dipole moment larger than in the crystal.

Utilization of monensin for detection of microdomains in cholesterol containing membrane.

The effect of cholesterol on the monensin mediated proton-cation exchange reaction was measured in the time-resolved domain. The experimental system consisted of a black lipid membrane equilibrated with monensin (Nachliel, E., Finkelstein, Y. and Gutman, M. (1996) Biochim. Biophys. Acta 1285, 131-145). The membrane separated two compartments containing electrolyte solutions and pyranine (8-hydroxypyrene 1,3,6-trisulfonate) was added on to one side of the membrane. A short laser pulse was used to cause a brief transient acidification of the pyranine-containing solution and the resulting electric signal, derived from proton-cation exchange, was measured in the microsecond time domain. Incorporation of cholesterol had a clear effect on the electric transients as measured with Na+ or K+ as transportable cations. The measured transients were subjected to rigorous analysis based on numeric integration of coupled, non-linear, differential rate equations which correspond with the perturbed multi-equilibria state between all reactants present in the system. The various kinetic parameters of the reaction and their dependence on the cholesterol content had been determined. On the basis of these observations we can draw the following conclusions: (1) Cholesterol perturbed the homogeneity of the membrane and microdomains were formed, having a composition that differed from the average value. The ionophore was found in domains which were practically depleted of phosphatidylserine. (2) The diffusivity of the protonated monensin (MoH) was not affected by the presence of cholesterol, indicating that the viscosity of the central layer of the membrane was unaltered. (3) The diffusivity of the monensin metal complexes (MoNa and MoK) was significantly increased upon addition of cholesterol. As the viscosity along the cross membranal diffusion route is unchanged, the enhanced motion of the MoNa and MoK is attributed to variations of the electrostatic potential within the domains.

Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane.

The dynamics of proton binding to the extracellular and the cytoplasmic surfaces of the purple membrane were measured by laser-induced proton pulses. Purple membranes, selectively labeled by fluorescein at Lys-129 of bacteriorhodopsin, were pulsed by protons released in the aqueous bulk from excited pyranine (8-hydroxy-1,3,6-pyrenetrisulfonate) and the reaction of protons with the indicators was measured. Kinetic analysis of the data imply that the two faces of the membrane differ in their buffer capacities and in their rates of interaction with bulk protons. The extracellular surface of the purple membrane contains one anionic proton binding site per protein molecule with pK = 5.1. This site is within a Coulomb cage radius (approximately 15 A) from Lys-129. The cytoplasmic surface of the purple membrane bears 4-5 protonable moieties (pK = 5.1) that, due to close proximity, function as a common proton binding site. The reaction of the proton with this cluster is at a very fast rate (3.10(10) M-1.s-1). The proximity between the elements is sufficiently high that even in 100 mM NaCl they still function as a cluster. Extraction of the chromophore retinal from the protein has a marked effect on the carboxylates of the cytoplasmic surface, and two to three of them assume positions that almost bar their reaction with bulk protons. The protonation dynamics determined at the surface of the purple membrane is of relevance both for the vectorial proton transport mechanism of bacteriorhodopsin and for energy coupling, not only in halobacteria, but also in complex chemiosmotic systems such as mitochondrial and thylakoid membranes.

Quantitative evaluation of the dynamics of proton transfer from photoactivated bacteriorhodopsin to the bulk.

It has been reported by many research groups that protons released during the photocycle of bacteriorhodopsin are detected by surface bound indicators much faster than by indicators in the bulk. In this study we used numerical simulation of chemical reaction's dynamics for analyzing the delayed appearance of protons in the bulk. The results indicate that the low pK surface groups of the membrane, which form an undilutable concentrated matrix of proton binding sites, retain the protons in this space according to the mass action law. The main sites for proton accumulation are the cluster of carboxylates on the cytoplasmic side of the membrane. The protonation of an indicator in the bulk does not proceed by its reaction with free proton, but rather through self-diffusion of the indicator to the membrane and abstraction of proton from the protonated surface group. The detailed mechanisms which correspond with these reactions are reported.

The dynamics of proton transfer at the C side of the mitochondrial membrane: picosecond and microsecond measurements.

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-trisulfonate), was introduced into the inner aqueous space of inside-out submitochondrial particles (SMP). Upon initiation of respiration, the dye recorded acidification of this space. Incorporation of high concentrations of the dye (approximately 100 nmol/mg of protein) had no effect on the respiratory functions of the vesicles, nor on their capacity to execute delta microH(+)-coupled reverse electron transfer. The respiratory control ratio (RCR) remained as high as RCR > 4. Pulse irradiation of the dye caused photodissociation of the proton from the 8-hydroxy position. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecond fluorimetry measured the dissociation of the proton from the excited dye molecule (phi OH*), while microsecond spectroscopy followed the reaction between the proton and the ground-state anion (phi O-). Numerical integration of the differential rate equations describes the diffusion of protons in the perturbed system. The nanosecond measurements yield the physical characteristics of the aqueous phase that dissolves the dye. The apparent dielectric constant of that space is rather low (epsilon = 20). The diffusion coefficient of the proton is 2.3 x 10(-5) cm2/s, and the activity of water is aH2O = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is taken up by the hydration layer of the lipids and proteins of the C side of the membrane. The microsecond dynamics measurements indicate that the rates of proton binding to the membrane surface components reach an equilibrium within 60 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of bulk protons with a mitochondrial inner membrane preparation: time-resolved measurements and their analysis.

The laser-induced proton pulse technique [Gutman, M. (1986) Methods Enzymol. 127, 522-538] was applied on suspensions of submitochondrial vesicles, and the exchange of protons between the bulk and the mitochondrial membranes was measured in the time-resolved domain with a submicrosecond resolution. The protons were discharged by photoexcitation of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) by a short laser pulse, and the reprotonation of the pyranine anion was monitored at 457.8 nm. In parallel, the protonation of the membrane was followed at 496.5 nm, looking at the transient absorbance of fluorescein, covalently attached to the M side of the membrane. The analysis of the relaxation dynamics was carried out by a simulation procedure that reconstructs the observed dynamics of the two chromophores. The analysis revealed the presence of the membrane indigenous buffering moieties. The low-pK buffer (pK 4.1) was present in a quantity of 100 +/- 20 nmol/mg of protein, and its kinetics indicate that it appears in multianionic clusters bearing a negative electric charge. The medium-pK buffer (pK 6.9) was present in a larger quantity (200 +/- 20 nmol/mg), and its kinetic parameter indicated clustering into positively charged domains. Both types of indigenous buffer reacted with the proton and pyranine anion in unhindered diffusion-controlled reactions. On the other hand, the exchange of protons between the indigenous buffer moieties was rather slow. No evidence was found for the presence of sites capable of retaining a proton, secluded from the bulk, for a time frame longer than 100 microseconds as required by the models of localized proton gradient.

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.

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.