Is there a weak H‐bond → LBHB transition on tetrahedral complex formation in serine proteases?

The transformation of a weak hydrogen bond in the free enzyme into a low-barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H-bond in the Asp102-His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self-consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102-His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme-inhibitor TC is a strong ionic asymmetric one-well hydrogen bond, in contrast to a previous suggestion that it is a weak H-bond in the former and a double-well LBHB in the latter.     

Polarized fluorescent emission from probes near dielectric interfaces

The electromagnetic field surrounding and emitted by a dipolar molecular probe very near to a dielectric interface is the sum of the real dipole field and the field of the image dipole induced inside the dielectric interface. The total charge distribution, made up of the real and image dipoles in close proximity to each other, approximates a quadrupole distribution and emits a light intensity pattern similar to that of an oscillating electric quadrupole. The electromagnetic field emitted by this system contains information that can be directly related to the spatial and orientational distributions of the dipole near the interface. Experimental methods are discussed that utilize this system for determining the spatial and orientational distribution of fluorescent probes in biological material.     

Calculation of isotope effects from first principles

Various means of calculating the effect of changing the mass of a given atom upon a chemical process are reviewed. Of particular interest is the deuterium isotope effect comparing the normal protium nucleus with its heavier deuterium congener. The replacement of the bridging protium in a neutral hydrogen bond such as the water dimer by a deuterium strengthens the interaction by a small amount via effects upon the vibrational energy. In an ionic H-bond such as the protonated water dimer, on the other hand, the reverse trend is observed in that replacement of the bridging protium by dimer weakens the interaction. In addition to the stability of a given complex, the rate at which a proton transfers from one group to another is likewise affected by deuterium substitution, viz. kinetic isotope effects (KIEs). The KIE is enlarged as the temperature drops, particularly so if the calculation of KIE includes proton tunneling. The KIE is also sensitive to any angular distortions or stretches present in the H-bond of interest. KIEs can be computed either by the standard transition state theory which is derived via only two points on the potential energy surface, or by more complete formalisms which take account of larger swaths of the surface. While more time intensive, the latter can also be applied to provide insights important in interpretation of experimental data.     

Classical Molecular Dynamics Simulation of Kappa Squared Factor in Resonance Energy Transfer for Linear Dipole Models

Molecular dynamics simulations of linear models interacting through a dipolar Kihara intermolecular potential are presented. Molecular orientation correlations are used to calculate the orientational factor kappa squared in the resonance energy transfer (RET) as a function of the intermolecular separation. The distance, R 0 (2/3), at which the simulated systems show an isotropic behavior is calculated and an analysis of the dependence of R 0 (2/3) on microscopic properties (molecular aspect ratio and dipole moment) as well on thermodynamics (temperature and density) is presented. An explanation of the use of metallic cations as probes in RET is given and some relations of our models with biological molecules are pointed out.     

An electrostatic model of a membrane ion pump

The model is based upon an ion channel with an electric dipolar structure. With simplifying assumptions it is possible to calculate that a typical channel, 1 nm in diameter and 5 nm long, could contain at most two or three univalent cations at a time. The channel ion binding sites have an effective affinity for ions from the fluid bathing the negative end of the channel, several orders of magnitude higher than their affinity for ions from the fluid bathing the positive end of the channel. The approach of an external, positively charged body to the negative end of the channel, is sufficient to convert the two- or three-channel ion sites with high affinity for ions from the fluid bathing this end into very low affinity sites for the same ions that now have access only to the fluid bathing the other end of the channel. The change in affinity and fluid access requires no molecular or electrical change in the channel structure other than the passive superposition of the electrostatic potential of the dipolar channel and that of the charged body. An oscillating electric field externally applied to an electric dipolar channel is shown to result in the unidirectional pumping of cations in the direction of the channel dipole even against large adverse ion concentration gradients. The energy required must be supplied by the sources of the electric field. By using two such channels in close proximity, one selective for K+ ions with its dipole moment pointing into a cell and the other selective for Na+ ions with its dipole moment pointing out from the cell, it is possible to construct a model pump with calculated properties that simulate many of those measured for Na+-K+-ATPase, with both physiological and artificial ionic concentrations.     

Stabilization of the electron in the quinone acceptor part of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> reaction centers

The evolution of the light-induced absorption difference spectrum (380–500 nm) of the reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides has been examined over 200 μs. The observed changes are interpreted as the effects of proton movement along the H-bond between the primary quinone acceptor and its protein surroundings. A theoretical analysis of the spectral evolution, considering the proton tunneling kinetics, corroborates this interpretation. The electronic state of the primary quinone is stabilized within tens of microseconds; the process is retarded upon deuteration of the reaction center as well as in 90% glycerol, and accelerated upon nondestructive heating to 40°C.     

Medium polarization effect on proton potential shape S.A SCRF-MO CNDO2 study of methanol and methanethiol H-bonded to imidazol

The theoretical calculations carried out with the self-consistent reaction field theory of solvent (medium) effects,have shown that the proton potential curve shapes of the above mentioned H-bonded systems are remarkably dependant upon the reaction field susceptibility parameter. The results show that the imidazol system would display different pK's at different sites of a given globular protein. Moreover, a proton translocation process is shown to be an essentially medium driven effect.     

Solution H NMR characterization of substrate-free C. diphtheriae heme oxygenase: Pertinence for determining magnetic axes in paramagnetic substrate complexes

Proton 2D NMR was used to confirm in solution a highly conserved portion of the molecular structure upon substrate loss for the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae, HmuO. The chemical shifts for the conserved portion of the structure are assessed as references for the dipolar shifts needed to determine the orientation of the paramagnetic susceptibility tensor, χ, in paramagnetic substrate complexes of HmuO. It is shown that the chemical shifts for the structurally conserved portion of substrate-free HmuO serve as excellent references for residues with only small to moderate sized dipolar shifts in the cyanide-inhibited substrate complex of HmuO, yielding an orientation of χ that is essentially the same as conventionally obtained from large dipolar shifts based on empirical estimates of the diamagnetic reference. The implications of these diamagnetic chemical shifts for characterizing the hydrogen bonding in the physiologically relevant, resting-state, high-spin aquo complex are discussed. The pattern of labile proton exchange in the distal H-bond network of substrate-free HmuO allowed comparison of changes in dynamic stability of tertiary contacts in the substrate-free and substrate-bound HmuO and with the same complexes of human heme oxygenase.     

Transformation of chemical energy in biomolecular systems with hydrogen bonds

The mechanism underlying the excitation of the hydrogen bond with ATP hydrolysis was considered. Coulomb interactions of the proton of the hydrogen bond A-H...B with the electrical field of the covalent bond of ADP-P were calculated. It was shown that the electrical field of the covalent bond of ADP-P excites oscillations of the proton in the complex with the hydrogen bond A-H...B and displaces it from the equilibrium towards the covalent bond. The distortion of the potential curve depends on a change in the length of the covalent bond of ADP-P. Adiabatic potentials U0 and UN of the ADP-P system were calculated, which correspond to the ground and excited states of the H-bond proton. It was found that as the length of the bond of ADP-P (rho) increases, the branches of the adiabatic potential U0(rho) and UN(rho) intersect. At the intersection point, the system can transit to the branch UN(rho), which can lead to a reduction of the barrier and a break of the covalent bond of ADP-P. Presumably, this mechanism is universal for processes of transformation of the chemical energy of ATP to the energy of excited hydrogen bond, a mechanism for the maintenance of heat balance and reduction of entropy in a living organism.     

Prototropic tautomerism of imidazolone in aqueous solution: a density functional approach using the combined discrete/self-consistent reaction field (SCRF) models

A systematic investigation of the proton transfer in the keto-amino/enol tautomerization of imidazolone was undertaken. Calculations in aqueous solution were performed using both combined discrete/self-consistent reaction field (SCRF) and SCRF methods. Complexes containing one to three water molecules around the hydrophilic site of imidazolone were used for the combined discrete/SCRF calculations. The DFT results predict that the barrier height for non-water-assisted intramolecular proton transfer is very high (214.8 kJmol^–1). Hydrogen bonding between imidazolone and the water molecule(s) will dramatically lower the barrier by a concerted multiple proton transfer mechanism. The proton transfer process through a eight-member ring formed by imidazolone and two water molecules is found to be more efficient and the calculated barrier height is ca. 61 kJmol^–1.Figure DFT calculations in aqueous solution predict the H-bonding between imidazolone(IZ) and the water molecule(s) will dramatically lower the tautomeric barrier by a concerted multiple proton transfer mechanism, in which an eight-member ring structure formed by IZ and 2H₂O is found to be more efficient and the barrier is 60.8 kJ mol^–1, much less than 214.8 kJ mol^–1 in the non-water-assisted mechanism.     

Influence of the redox potential of the medium on the activity of polyphenol oxidase

While ascorbate is an inhibitor of polyphenol oxidase upon its activity on tyrosine, hydrogen peroxide has a promoting effect especially in conjunction with catalase. When both ascorbate and peroxide are present, the effect of the former dominates over the latter. These influences can be explained on the basis of the redox potential of the solutions: high potentials promote, while low potentials decrease the rate of the oxidative enzyme reaction. The findings might have some implications on the process of aging of tissues.     

Electric birefringence of eukaryotic ribosomes

Eukaryotic 60S ribosomal subunits were studied by transient electric birefringence. Conformations of subunits in active and inactive states upon changes in magnesium concentration were compared by electric birefringence and orientational relaxation time measurements. Active subunits exhibit a positive birefringence and a relaxation time of the order of 8 microseconds. In the presence of EDTA, inactive subunits show no birefringence. When Mg2+ is reverted in the cold to its initial level, the electro-optical properties of the subunits are partially restored although the particles remain biologically inactive.     

The contribution of proton fluctuation to dielectric relaxation in protein solutions

Equations are derived which account for the effect of an applied electric field on the fluctuation of protons associated with a macromolecule. The contribution of this proton polarization to the complex permittivity of the macromolecules is evaluated in terms of its effect on both the dispersion due to proton fluctuation and the dispersion caused by dipolar rotation. An expression for the fluctuation correlation time τiδ is derived in terms of the mean lifetimes of the ionised and unionised state τ0 and τ+. If τiδ is very much less than the dipolar correlation time τi, the fluctuation dispersion will occur at much higher frequencies than the dispersion due to orientation polarization; hence the dispersions will be well separated. If τiδ » τi the two regions would overlap and would be indistinguishable as separate entities. At present insufficient data are available to test rigorously these conclusions but the potentialities of the theory in relation to small globular proteins is shown.     

A theory of charge separation,ion, electron and proton transport in photosynthetic membranes based on asymmetry of surface charges

We present a model for the light-induced charge separation, proton and ion transport across photosynthetic membranes based on an assumption of the transmembrane surface charge asymmetry. In dark equilibrium, this asymmetry gives rise to an internal membrane electric field whose direction is perpendicular to the membrane surfaces. The role of the field in the light-induced charge separation is similar to the function of the built-in electric field across a solid-state p-n junction. Light-generated free charge carriers in the membrane flow according to its direction and upon recombination on the surface give rise to an electrochemical potential difference for electrons across the membrane. The associated coupled electron-proton transport, and ion diffusion can be viewed as a response of the system to the light-induced redox and electric potential changes.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. The quenching effect of blue light

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M₄₁₂) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

NMR approaches for monitoring domain orientations in calcium-binding proteins in solution using partial replacement of Ca2+ by Tb3+.

This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 A from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains.     

Nerve membrane electrical characteristics near the resting state

Summary An experimental analysis of the squid axon membrane impedance in the vicinity of the resting state and as a function of frequency is presented. Particular attention was devoted to the measurement of theresonance frequency, for which the absolute magnitude of the impedance attains its maximum value, in different, extracellular solutions, at various temperatures and in the presence of constant depolarizations or hyperpolarizations.The variations in the concentration of sodium, potassium and divalent ions and the addition of tetrodotoxin, changed markedly the maximum impedance but had little effect, at a fixed temperature, on the resonance frequency, whose temperature dependance is described by aQ ₁₀ variable from 3.7 (around 4 °C) to 1.9 (around 15 °C). Substitution of heavy water decreased the resonance frequency by a factor 1.25, fairly independent of temperature. Steady depolarizations or hyperpolarizations produced large variations of the resonance frequency, with strong temperature dependance.The results indicate that the resonance frequency is directly related to the membrane permeability changes which take place quite independently of the composition of the extra cellular solution and are governed by the electric field existing within the membrane structure rather than by the total membrane potential, to which membrane-solution boundary potentials can give a large contribution.     

Evidence for electrogenic proton extrusion by subepidermal cells of Lemna paucicostata 6746

The cell potential of Lemna paucicostata 6746 was measured between the vacuole and the external solution. The potential in the dark (-202 mV) could be depolarized with 0.1 mM dicyclohexyl carbodiimide (DCCD) or 1 mM arsenate to-81 mV. The hyperpolarization above the latter value is therefore attributed to an ATP-dependent process. The cell potential showed a significant dependence upon the pH of the external solution. The change in the potential induced by a jump in pH between two certain values, was reversible and independent of the mode of performing the pH change (stepwise or at once). The DCCD-or arsenate-depolarized potential did not respond to external pH changes. A 0.1 mM ammonium chloride solution depolarized the cell potential reversibly to-83 mV. This potential-change could be greatly reduced by simultaneous addition of 5 mM Na isobutyrate. The pH sensitivity of the cell potential is ascribed to changes in the rate of proton extrusion upon altering the proton gradient across the plasmalemma. The effects of ammonium and isobutyrate are interpreted as being the consequence of pH shifts at the inner face of the plasmalemma, caused by the permeation of the undissociated form of the weak acid or base. A critical discussion of an alternative interpretation for the ammonium effect is presented.Abbreviation DCCD N,N-dicyclohexyl carbodiimide