pH-dependent redox potential: how to use it correctly in the activation energy analysis

The activation barrier (the activation free energy) for the reaction's elementary act proper does not depend on the presence of reactants outside the reaction complex. The barrier is determined directly by the concentration-independent configurational free energy. In the case of redox reactants with pH-dependent redox potential, only the pH-independent quantity, the configurational redox potential enters immediately into expression for activation energy. Some typical cases of such reactions have been discussed (e.g., simultaneous proton and electron detachment, acid dissociation followed by oxidation, dissociation after oxidation, and others). For these mechanisms, the algorithms for calculation of the configurational redox potential from the experimentally determined redox potentials have been described both for the data related to a dissolved reactant or to a prosthetic group of an enzyme. Some examples of pH-dependent enzymatic redox reactions, in particular for the Rieske iron-sulfur protein, have been discussed.     

The relationship between ligand-binding thermodynamics and protein-ligand interaction forces measured by atomic force microscopy.

The interaction forces between biotin and a set of streptavidin site-directed mutants with altered biotin-binding equilibrium and activation thermodynamics have been measured by atomic force microscopy. The AFM technique readily discriminates differences in interaction force between the site-directed (Trp to Phe or Ala) mutants. The interaction force is poorly correlated with both the equilibrium free energy of biotin binding and the activation free energy barrier to dissociation of the biotin-streptavidin complex. The interaction force is generally well correlated with the equilibrium biotin-binding enthalpy as well as the enthalpic activation barrier, but in the one mutant where these two parameters are altered in opposite directions, the interaction force is clearly correlated with the activation enthalpy of dissociation. These results suggest that the AFM force measurements directly probe the enthalpic activation barrier to ligand dissociation.     

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.     

Radical formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics: the reactivity of these radicals with GSH, DNA, and unsaturated lipid

Radicals generated by the peroxidase catalyzed oxidation of a wide variety of substrates oxidize GSH, NADH, or arachidonate with accompanying oxygen activation. Substrates studied include carcinogens, drugs, or xenobiotics. The effectiveness of the various radicals is partly related to their one-electron oxidation potential. High redox potential radicals were particularly effective at oxidizing these biomolecules. Low redox potential radicals did not react with GSH, NADH, or arachidonate, but can directly activate oxygen to form hydroxyl radicals or undergo scission to carbon radicals. The hydroxyl and carbon radicals have a high redox potential and readily oxidize biomolecules. DNA strand breakage also occurs with some high redox potential radicals, but DNA did not react with low redox potential radicals. The extensive binding of xenobiotics to DNA in the peroxidase system was attributed to noncovalent binding by polymeric products or covalent binding by the two electron oxidation product (formed by radical dismutation or oxidation). The latter can cause alkali labile DNA strand breaks. GSH conjugate formation was also attributed to the two electron oxidation product. Radicals have been trapped in intact cells and oxygen activation or lipid peroxidation has been demonstrated but it is still not clear whether the associated GSH oxidation, DNA strand breakage and cytotoxicity is the result of direct action by radicals. Indirect enzymic mechanisms for free radical mediated DNA strand breakage and cytotoxicity are discussed.     

A stopped-flow study of the reaction of reduced cytochrome oxidase with oxygen

The reaction of a reduced cytochrome oxidase system consisting of beef heart cytochrome oxidase, cytochrome c, and ascorbate with molecular oxygen was kinetically and thermodynamically investigated using a stopped-flow, rapid wavelength-scanning technique. Processes for oxidation of ferrocytochrome a, bound ferrocytochrome c, and free ferrocytochrome c have been identified, and their rate constants have been determined. Values of the activation energy for these reactions indicate that the oxidation of bound ferrocytochrome c is a simple chemical electron-transfer process and that oxidations of ferrocytochrome a and free ferrocytochrome c are complex processes involving changes in protein conformation.     

Different mechanisms of free fatty acid flip-flop and dissociation revealed by temperature and molecular species dependence of transport across lipid vesicles

The mechanism of free fatty acid (FFA) transport across membranes is a subject of intense investigation. We have demonstrated recently that flip-flop is the rate-limiting step for transport of oleic acid across phospholipid vesicles (Cupp, D., Kampf, J. P., and Kleinfeld, A. M. (2004) Biochemistry 43, 4473-4481). To better understand the nature of the flip-flop barrier, we measured the temperature dependence of a series of saturated and monounsaturated FFA. We determined the rate constants for flip-flop and dissociation for small (SUV), large (LUV), and giant (GUV) unilamellar vesicles composed of egg phosphatidylcholine. For all FFA and vesicle types, dissociation was faster than flip-flop, and for all FFA, flip-flop and dissociation were faster in SUV than in LUV or GUV. Rate constants for both flip-flop and dissociation decreased exponentially with increasing FFA size. However, only the flip-flop rate constants increased significantly with temperature; the barrier to flip-flop was virtually entirely due to an enthalpic activation free energy. The barrier to dissociation was primarily entropic. Analysis in terms of a simple free volume (V(f)) model revealed V(f) values for flip-flop that ranged between approximately 12 and 15 Angstroms(3), with larger values for SUV than for LUV or GUV. V(f) values increased with temperature, and this temperature dependence generated the enthalpic barrier to flip-flop. The barrier for dissociation and its size dependence primarily reflect the aqueous solubility of FFA. These are the first results to distinguish the energetics of flipflop and dissociation. This should lead to a better understanding of the mechanisms governing FFA transport across biological membranes.     

Electrostatic and redox potential effects on the rat of electron-transfer reaction of nicotinamide adenine dinucleotides with 1-substituted 5-ethylphenazines

The effects of redox potential and electric charge on the rate of electron-transfer reaction by a two-electron process were investigated. For electron donors, beta-NADH, beta-NADPH and alpha-NADH were used; they have similar structures but different charges and different redox potentials. For electron acceptors, the following 5-ethylphenazine derivatives were used: 1-(3-carboxypropyloxy)-5-ethylphenazine, 1-(3-ethoxycarbonylpropyloxy)-5-ethylphenazine, and 1-[N-(2-aminoethyl)carbamoylpropyloxy]-5-ethylphenazine. They have similar structures and different charges. Using these donors and acceptors, the potential and the charge effects were estimated separately. In the potential effect, a linear free energy relationship was observed for the change in the redox potential of the donor with a Br?nsted slope of about unity. On the other hand, the slope for the change in the potential of the acceptor was about 0.5. These results show that the potential effect due to electron donors is different from that due to electron acceptors. A linear relationship was also observed between activation free energy and electrostatic force (or potential). The redox potential effect and the electrostatic effect are independent and additive. New theory for the mechanism of electron-transfer reactions is needed to explain these results.     

Oxyl radicals, redox-sensitive signalling cascades and antioxidants

Oxidative stress is an increase in the reduction potential or a large decrease in the reducing capacity of the cellular redox couples. A particularly destructive aspect of oxidative stress is the production of reactive oxygen species (ROS), which include free radicals and peroxides. Some of the less reactive of these species can be converted by oxidoreduction reactions with transition metals into more aggressive radical species that can cause extensive cellular damage. In animals, ROS may influence cell proliferation, cell death (either apoptosis or necrosis) and the expression of genes, and may be involved in the activation of several signalling pathways, activating cell signalling cascades, such as those involving mitogen-activated protein kinases. Most of these oxygen-derived species are produced at a low level by normal aerobic metabolism and the damage they cause to cells is constantly repaired. The cellular redox environment is preserved by enzymes and antioxidants that maintain the reduced state through a constant input of metabolic energy. This review summarizes current studies that have been regarding the production of ROS and the general redox-sensitive targets of cell signalling cascades.     

Impact of enzyme motion on activity

Experimental and theoretical data imply that enzyme motion plays an important role in enzymatic reactions. Enzyme motion can influence both the activation free energy barrier and the degree of barrier recrossing. A hybrid theoretical approach has been developed for the investigation of the relation between enzyme motion and activity. This approach includes both electronic and nuclear quantum effects. It distinguishes between thermally averaged promoting motions that influence the activation free energy barrier and dynamical motions that influence the barrier recrossings. Applications to hydride transfer in liver alcohol dehydrogenase and dihydrofolate reductase resulted in the identification and characterization of important enzyme motions. These applications have also led to the proposal of a network of coupled promoting motions in enzymatic reactions. These concepts have important implications for protein engineering and drug design.     

Redox and addition chemistry of quinoid compounds and its biological implications

The overall biological activity of quinones is a function of the physico-chemical properties of these compounds, which manifest themselves in a critical bimolecular reaction with bioconstituents. Attempts have been made to characterize this bimolecular reaction as a function of the redox properties of quinones in relation to hydrophobic or hydrophilic environments. The inborn physico-chemical properties of quinones are discussed on the basis of their reduction potential and dissociation constants, as well as the effect of environmental factors on these properties. Emphasis is given on the effect of methyl-, methoxy-, hydroxy-, and glutathionyl substituents on the reduction potential of quinones and the subsequent electron transfer processes. The redox chemistry of quinoid compounds is surveyed in terms of a) reactions involving only electron transfer, as those accomplished during the enzymic reduction of quinones and the non-enzymic interaction with redox couples generating semiquinones, and b) nucleophilic addition reactions. The addition of nucleophiles, entailing either oxidation or reduction of the quinone, are exemplified in reactions with oxygen- or sulfur nucleophiles, respectively. The former yields quinone epoxides, whereas the latter yields thioether-hydroquinone adducts as primary molecular products. The subsequent chemistry of these products is examined in terms of enzymic reduction, autoxidation, cross-oxidation, disproportionation, and free radical interactions. The detailed chemical mechanisms by which quinoid compounds exert cytotoxic, mutagenic and carcinogenic effects are considered individually in relation to redox cycling, alterations of thiol balance and Ca++ homeostasis, and covalent binding.     

Analysis of the pH-dependencies of the association and dissociation kinetics of HIV-1 protease inhibitors

The kinetic constants for the interactions between HIV-1 protease and a selection of inhibitors were determined at different pH-values using a biosensor based interaction assay. Since this technique does not involve a substrate, it was possible to determine the pH-dependencies of the association and dissociation rates of an inhibitor, without the complication of a pH-dependent enzyme-substrate/product equilibrium. The importance of these interactions was evaluated by correlating the free energy changes upon association and dissociation of inhibitors with the predicted change in electrostatic properties of the interacting groups as a result of altered pH. It was found that the kinetic parameters varied with pH in a unique manner for all inhibitors, demonstrating that the kinetic features were associated with the specific structure of each inhibitor. Association and dissociation had different pH-profiles, indicating that the two processes proceeded by different pathways/mechanisms. The energy barrier for dissociation of the enzyme-indinavir complex increased with pH from 4.1 to 7.4, while it was generally reduced for the other inhibitors as the pH was increased from 5.1 to 7.4. The pH-dependent interactions involved in the recognition/binding of inhibitors and in the stabilization of the complex were identified by analysing three-dimensional structures of enzyme-inhibitor complexes. The interaction between the pyridine nitrogen of indinavir with Arg-8 was hypothesized to be responsible for the unique pH-dependency of indinavir. The analysis revealed features of interactions that are significant for understanding enzyme function and for optimization of new drug leads. It also highlighted the importance of environmental conditions on interactions.     

Electron transfer between flavodoxin semiquinone and c-type cytochromes: correlations between electrostatically corrected rate constants, redox potentials, and surface topologies

We have measured the ionic strength dependence of the rate constants for electron transfer from the semiquinone of Clostridium pasteurianum flavodoxin to 12 c-type cytochromes and several inorganic oxidants using stopped-flow methodology. The experimental data were fit quite well by an electrostatic model that represents the interaction domains as parallel disks with a point charge equal to the charge within this region of the protein. The analysis provides an evaluation of the electrostatic interaction energy and the rate constant at infinite ionic strength (k affinity). The electrostatic charge on the oxidant within the interaction site can be obtained from the electrostatic energy, and for most of those reactants for which structures are available, the results are in good agreement with expectation. The k affinity values were found to correlate with redox potential differences, as expected from the theory of adiabatic (or nonadiabatic) outer-sphere electron-transfer reactions. Deviations from the theoretical curves are interpreted in terms of the influence of surface topology on reaction rate constants. In general, we find that electrostatic effects, steric influences, and redox potential all exert a much larger effect on reaction rate constants for the flavodoxin-cytochrome system than has been previously observed for free flavin-cytochrome interactions. The implications of this for determining biological specificity are discussed.     

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.     

Reaction pathway and free energy profiles for butyrylcholinesterase-catalyzed hydrolysis of acetylthiocholine

The catalytic mechanism for butyrylcholineserase (BChE)-catalyzed hydrolysis of acetylthiocholine (ATCh) has been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy (QM/MM-FE) calculations on both acylation and deacylation of BChE. Additional quantum mechanical (QM) calculations have been carried out, along with the QM/MM-FE calculations, to understand the known substrate activation effect on the enzymatic hydrolysis of ATCh. It has been shown that the acylation of BChE with ATCh consists of two reaction steps including the nucleophilic attack on the carbonyl carbon of ATCh and the dissociation of thiocholine ester. The deacylation stage includes nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain. QM/MM-FE calculation results reveal that the acylation of BChE is rate-determining. It has also been demonstrated that an additional substrate molecule binding to the peripheral anionic site (PAS) of BChE is responsible for the substrate activation effect. In the presence of this additional substrate molecule at PAS, the calculated free energy barrier for the acylation stage (rate-determining step) is decreased by ~1.7 kcal/mol. All of our computational predictions are consistent with available experimental kinetic data. The overall free energy barriers calculated for BChE-catalyzed hydrolysis of ATCh at regular hydrolysis phase and substrate activation phase are ~13.6 and ~11.9 kcal/mol, respectively, which are in reasonable agreement with the corresponding experimentally derived activation free energies of 14.0 kcal/mol (for regular hydrolysis phase) and 13.5 kcal/mol (for substrate activation phase).     

Membrane catalysis: synchronous multielectron reactions at the interface between two liquid phases. Bioenergetic mechanisms

Kinetics of multi-electron reactions at the interface between two immiscible liquids are considered. Calculations of the energy of solvent reorganization, of the work required to bring reactants and reaction products together, and of the electrostatic contributions to the Gibbs free energy of the reaction during electron transfer between reactants which are in different dielectric media are reported. Conditions under which the free energy of activation of the interfacial reaction of electron transfer decreases are established. The influence of the distance between reactants and of the dielectric permittivity of the non-aqueous phase on the solvent reorganization energy value is studied. Conditions under which multielectron reactions at the interface proceed are discussed. The biophysics and biochemistry of photosynthesis and respiration are considered as examples of multielectron processes.     

Energetics for oxidation of a bound manganese cofactor in modified bacterial reaction centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn2+ with a dissociation constant of 1 μM at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P+ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn2+ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P+. The extent of P+ reduction was found to systematically range from a full reduction in the mutants with high P/P+ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn2+ oxidation on the P/P+ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn2+/Mn3+ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn2+/Mn3+ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn2+ oxidation ranging from 30 to 120 s(-1) as the P/P+ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s(-1), reflecting an increase in the free energy difference due to the lower Mn2+/Mn3+ midpoint potential. This dependence of the rate constant on the P/P+ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s(-1) and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn4Ca cofactor of photosystem II.     

Short-lived delayed luminescence of photosynthetic organisms. I. Nanosecond afterglows in purple bacteria at low redox potentials.

A combined study of emissions of purple bacteria Rhodospirillum rubrum, Ectothiorhodospira shaposhnikovii and Thiocapsa roseopersicina was performed under conditions of low potential. It has been shown that a considerable part of the emission represents a delayed luminescence with a lifetime of about 5 ns and an activation energy delta E = 0.05 +/- 0.03 eV. Intensity of this delayed luminescence is approximately equal to that of prompt fluorescence. It diminishes as temperature decreases and also as the intermediate acceptor I becomes reduced after prolonged illumination under low potential conditions. This luminescence represents a radiative decay of the intermediate state, PF, and the luminescence activation energy, delta E, reflects the energy barrier between P*-890 and PF. The value of this barrier determined in the present work is much lower than those obtained previously [3,4,26] for the free-energy release during the primary act of charge separation, basing on redox potential techniques. The reason for this discrepancy is discussed. Delayed luminescence in the picosecond time range is predicted to exist under conditions of active photosynthesis as a result of a small (approx. 0.05 eV) energy barrier between PF and the excited singlet state of reaction center bacteriochlorophyll.     

Cellular redox potential and the biomolecular electrochemical series: a systems hypothesis

The role of cellular redox potential in the regulation of protein activity is becoming increasingly appreciated and characterized. In this paper we put forward a new hypothesis relating to redox regulation of cellular physiology. We have exemplified our hypothesis using apoptosis since its redox phenomenology is well established, but believe that it is equally applicable to several other pathways. Our hypothesis is that since multiple proteins in the apoptosis pathway are thought to be regulated via oxidation/reduction reactions and since cellular redox potentials have been shown to become progressively more oxidative during apoptosis, that the proteins could be arranged in an electrochemical series where the protein's standard potential correlates with its position in the pathway. Since the most stable oxidation state of the protein is determined by its standard potential and the redox potential of its environment (in a way predictable by the Nernst equation), a quantitative model of the redox regulation of the pathway could be developed. We have outlined our hypothesis, illustrating it using a pathway map which assembles a selection of the literature on apoptosis into a readable graphical format. We have also outlined experimental approaches suitable for testing our hypothesis.     

A theoretical approach to the link between oxidoreductions and pyrite formation in the early stage of evolution

There are two fundamental axioms of surface metabolism theory: (i) pyrite formation from H2S and FeS is proposed as a source of energy for life, and (ii) archaic reductive citric acid cycle is put into the center of a metabolic network. However, the concept fails to indicate how sulfide oxidation ought to be coupled to processes driven by free energy change occurring during pyrite production, and secondly, how reductive citric acid cycle ought to be supplied with row material(s). Recently, the non-enzymatic methylglyoxalase pathway has been recommended as the anaplerotic route for the reductive citric acid cycle. In this paper a mechanism is proposed by which the oxidation of lactate, the essential step of the anaplerotic path, becomes possible and a coupling system between sulfide oxidation and endergonic reaction(s) is also presented. Oxidoreduction for other redox pairs is discussed too. It is concluded that the S(o)/H2S system may have been the clue to energy production at the early stage of evolution, as hydrogen sulfide produced by the metabolic network may have functioned as a coupling molecule between endergonic and exergonic reactions.     

Protection of an unstable reaction intermediate examined with linear free energy relationships in tyrosyl-tRNA synthetase

Linear free energy relationships (LFERs) are powerful tools in the search to understand the relationship between molecular structure and activity. They frequently link the changes in the rate constants for a reaction to changes in the equilibrium constant caused by alterations in structure. In physical-organic chemistry, these have been interpreted to give information on the structure of the transition state. Similar phenomena have been observed for reactions catalyzed by a series of engineered mutants of tyrosyl-tRNA synthetase from Bacillus stearothermophilus. LFERs are applied in this study to probe how the enzyme minimizes its side reactions. A linear free energy relationship is shown between the binding of the unstable enzyme-tyrosyl adenylate complex and its rate constant of hydrolysis. However, mutations of a key residue, His48, show significant deviation from the relationship, implying a role for the side chain in protection of the complex from hydroxide attack. A second linear free energy relationship is shown linking the rate and equilibrium constants for tyrosyl adenylate binding to the enzyme. Four distinct classes of mutation are discussed in the context of this relationship. The data from all but one of these groups of mutations conform well to a linear free energy relationship between the dissociation rate and dissociation equilibrium constants for the enzyme-tyrosyl adenylate complex with slope beta = 1.01 +/- 0.08. The specificity of binding of tyrosyl adenylate is determined solely by its dissociation rate constant of the intermediate, and the mutations have relatively little effect on the enzyme-tyrosyl adenylate association rate.(ABSTRACT TRUNCATED AT 250 WORDS)