A critique of the chemosmotic model of energy coupling.

The chemosmotic model provides a framework for visualizing energy-coupled reactions (vectorial reaction sequences, membrane-dependent gradient formation, and charge separation of reacting species) and a mechanism for energy coupling (indirect coupling between the driving and driven reaction sequences mediated by a membrane potential or a protonmotive force). The mechanistic parameters of this model have been examined from four standpoints: compatibility with the experimental realities, supporting evidence that is unambiguous, compatibility with the enzymic nature of energy coupling, and the capability for generating verifiable predictions. Recent developments that have clarified the mechanism of ion transport, the nature of the protonic changes that accompany energy coupling, and the enzymic nature of energy coupling systems have made such an examination both timely and necessary. After weighing the available evidence, it has been concluded that the chemosmotic principle of indirect coupling has no basis in fact and that it is physically unsound in respect to the mechanism of energy coupling and enzymic catalysis.     

Relation between enzymic catalysis and energy coupling.

The principles that underlie enzyme catalysis also apply to energy coupling processes. A comparison is made between a kinase system that mediates the phosphorylation of glucose by ATP (hexokinase), as the prototype for enzymic catalysis, and the mitochondrial electron-transfer complexes, as the prototypes for energy coupling systems. Induced polarization of chemical bonds and charge separation and elimination are common component events of both enzyme catalysis and energy coupling. Thus, definite limits can be imposed on models of energy coupling; they must comply with the basic principles of enzymic catalysis.     

Paired Moving Charges in Mitochondrial Energy Coupling

A model of mitochondrial energy coupling has been proposed based on the principles of paired charge separation and vectorial paired charge flow. The unique role of the electron transfer chain and ionophores in mediating charge separation is emphasized.     

Paired moving charge model of energy coupling. III. Intrinsic ionophores in energy coupling systems.

The experimental basis for the postulated role of intrinsic ionophores in mitochondrial ion transport and energy coupling is summarized. Intrinsic ionophores appear to be linked to, or contained within, specific ionophoroproteins localized in the inner membrane, and the isolation of these ionophores requires their release from the ionophoroproteins. At least ten different species of ionophores have been isolated from the mitochondrion, five of which have been wholly or in part chemically identified. Intrinsic ionophores have been implicated in the activation of inorganic phosphate in ATP synthesis and hydrolysis, and in the contol of the coupling modes. The presence of ionophores in soluble proteins such as troponin and in ATP-energized kinases has been demonstrated.     

Universal energy principle of biological systems and the unity of bioenergetics.

Electronic energy (chemical bond energy) is the exclusive source of utilizable energy in biological systems. The release of this energy is mediated enzymically. The energy required to rupture a single covalent or ionic bond is prohibitively high under physiological conditions [in the range of 80-200 kcal/mol (1 kcal = 4.18 kJ)]. By the technique of the pairing of bond rupture (two juxtaposed bonds ruptured simultaneously) and the pairing of bond formation, enzymes can bypass the huge thermodynamic barrier to chemical change inherent in rupture of a single bond and operate within thermal limits. Enzymes accordingly can be conceived of as the energy machines that translate this principle. The principle of this transduction is that the energy required for forming a new covalent bond can fall within thermal limits when the original charged atom partner to the bond is displaced by a substitute charged atom under conditions in which the charge field of the bond remains constant during the substitution. In the transition from classical enzymology to energy coupling, muscular contraction, template-dependent replication, etc., new dimensions and possibilities are added to the basic enzymatic machinery. Specialized molecular devices (membranes, filaments, channels, templates, etc.) have to be introduced to make possible these extensions and permutations of enzymology. But it is demonstrable that the basis pairing principle is fully preserved during any of these modifications or extensions. Long range movement--of an ion, a filament, or a template--is the most important property introduced into classical enzymology in the transition to energy coupling systems.     

Ion-transport chain of cytochrome oxidase: the two chain-direct coupling principle of energy coupling.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) couples the aerobic oxidation of ferrocytochrome c to the cyclical transport of monovalent cations or to the active transport of monovalent and divalent cations. This transport capability is mediated by an intracomplex ion-transport chain of two protein-bound molecules of cardiolipin per molecule of cytochrome oxidase. Cardiolipin in a two-phase system shows the identical ionophoric pattern as does the cytochrome oxidase coupled system. A molecular model of the cardiolipin chain suggests the possibility of a cage-like structure through which cations can be transferred from phosphate group to phosphate group. The ion-transport chain and the electron-transport chain are anchored to the same set of subunits (I+IV); the close proximity of the two chains argues for the direct coupling of electron and cation flow. The ion-transport chain of cytochrome oxidase provides an introduction to the molecular mechanisms by which ions are moved across membranes in energy-coupling systems.     

Predicting coupling limits from an experimentally determined energy landscape

Repeat proteins are composed of tandem structural modules in which close contacts do not extend beyond adjacent repeats. Despite the local nature of these close contacts, repeat proteins often unfold as a single, highly coupled unit. Previous studies on the Notch ankyrin domain suggest that this lack of equilibrium unfolding intermediates results both from stabilizing interfaces between each repeat and from a roughly uniform distribution of stability across the folding energy landscape. To investigate this idea, we have generated 15 variants of the Notch ankyrin domain with single and multiple destabilizing substitutions that make the energy landscape uneven. By applying a free energy additivity analysis to these variants, we quantified the destabilization threshold over which repeats 6 and 7 decouple from repeats 1-5. The free energy coupling limit suggested by this additivity analysis ( approximately 4 kcal/mol) is also reflected in m-value analysis and in differences among equilibrium unfolding transitions as monitored by CD versus fluorescence for all 15 variants. All of these observations are quantitatively predicted by analyzing the response of the experimentally determined energy landscape to increasing unevenness. These results highlight the importance of a uniform distribution of local stability in achieving cooperative unfolding.     

Rhythmogenic effects of weak electrotonic coupling in neuronal models.

Strong gap-junctional coupling can synchronize the electrical oscillations of cells, but we show, in a theoretical model, that weak coupling can phase lock two cells 180 degrees out-of-phase. Antiphase oscillations can exist in parameter regimens where in-phase oscillations break down. Some consequences are (i) coupling two excitable cells leads to pacemaking, (ii) coupling two pacemaker cells leads to bursting, and (iii) coupling two bursters increases burst period. The latter shows that details of the fast spikes can affect macroscopic properties of the slow bursts. These effects hold in other models for bursting and may play a role in the collective behavior of cellular ensembles.     

Stochastic free energy transduction.

Theoretical free-energy coupling systems in which the free energy coupling intermediate (e.g., the proton) occurs only in small numbers of molecules per coupling unit are shown to exhibit a number of peculiar properties: (i) the reactions involving the intermediates do not follow conventional kinetic (or nonequilibrium thermodynamic) rate laws in terms of the average concentration or chemical potential of the intermediate, (ii) the variation of the output reaction rate with the average intermediate concentration (or apparent chemical potential) is not unequivocal but depends on whether the input reaction or the leak is varied to alter that concentration, and (iii) when the apparent free energy contained in the average concentration of the intermediate is compared with the average free energy recovered in the output reaction, apparent violations of the second law of thermodynamics can occur. These phenomena are reminiscent of experimental observations in proton-linked free-energy transducing systems that suggest a more direct coupling between electron transfer chains and H+-ATPases than only through a bulk proton gradient, delta muH. Consequently, the chemiosmotic coupling theory can account for those observations if it limits the number of free energy coupling protons per chemiosmotic coupling unit to small values.     

Defibrillator electrode-chest wall coupling agents: influence on transthoracic impedance and shock success

The purpose of this study was to determine if the difference in transthoracic impedance produced by different coupling agents affects the success of shocks for defibrillation. Three different coupling agents, Harco pads (Hewlett-Packard), Littman pads (3M) and Redux paste (Hewlett-Packard), were assessed in 10 anesthetized dogs in which ventricular fibrillation was induced by electrical stimulation of the right ventricle. Defibrillation was attempted 15 seconds later, using 50, 100 and 150 joules (selected energy). Actual delivered energy, current, impedance and the percent of the shocks that achieved defibrillation were determined for the three coupling agents. Redux paste gave significantly lower impedance and higher current than the two disposable performed coupling pads tested. Despite this, there were no significant differences in shock success among the three coupling agents. Thus, in this experimental model, over a three-fold energy range, disposable coupling pads were as effective as electrode paste for defibrillation despite the slightly higher impedance of the disposable pads.     

Charge movement and depolarization-contraction coupling in arthropod vs. vertebrate skeletal muscle.

Voltage-dependent charge movement has been characterized in arthropod skeletal muscle. Charge movement in scorpion (Centuroides sculpturatus) muscle is distinguishable from that in vertebrate skeletal muscle by criteria of kinetics, voltage dependence, and pharmacology. The function of scorpion charge movement is gating of calcium channels in the sarcolemma, and depolarization-contraction coupling relies on calcium influx through these channels.     

Role of coupling entropy in establishing the nature and magnitude of allosteric response.

The coupling free energy between an allosteric ligand and a substrate, delta Gax, is an explicit measure of the nature as well as the magnitude of impact that an allosteric ligand has on the binding of the substrate ligand to the enzyme, with positive values indicating inhibition and negative values indicating activation. By measuring the variation with temperature of the coupling free energy between the allosteric ligand and the substrate, it is possible to determine the enthalpic and entropic components that give rise to the coupling free energy. We have performed this analysis on two different K-type allosteric systems: the allosteric inhibition of rat liver phosphofructokinase by MgATP, and the allosteric activation of beef heart NAD+-dependent isocitrate dehydrogenase by ADP. In both cases the coupling free energy arises as the net result of opposing enthalpic and entropic components, with the coupling enthalpy (delta Hax) favoring activation and the coupling entropy (delta Sax) favoring inhibition. For phosphofructokinase at 25 degrees C, the absolute value of T delta Sax is greater than the absolute value of delta Hax, and net inhibition of rat liver phosphofructokinase by MgATP is realized. For isocitrate dehydrogenase, delta Hax dominates; however, the net activation is substantially mitigated by the magnitude of T delta Sax. Hence, the coupling entropy plays an important role in establishing both the nature and magnitude of the allosteric response. We hypothesize that the negative coupling entropy arises from the particular constraint placed upon the internal dynamical properties of the enzyme by the simultaneous binding of both allosteric and substrate ligands.     

A thermodynamic coupling mechanism for GroEL-mediated unfolding.

Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.     

A cause of paired ventricular extrasystoles.

Eight patients with ventricular extrasystoles are reported in whom coupling intervals of the extrasystoles to the proceding sinus beats were variable and in whom paired ventricular extrasystoles were occasionally seen. In all patients, paired ventricular extrasystoles were initiated only by comparatively late coupled ventricular extrasystoles. However, the interval between the first and the second of these paired extrasystoles was always much shorter than the coupling interval of this first extrasystole to the preceding sinus beat, so that the latter extrasystole often interrupted the T wave of the first one, indicating the R-on-T phenomenon. In two patients there was a gap between the ranges of coupling intervals for single extrasystoles and for the first ones of paired extrasystoles. These observations suggest the presence of longitudinal dissociation in the reentrant pathway as one of the causes of paired ventricular extrasystoles.     

Photoisomerization, energy storage, and charge separation: A model for light energy transduction in visual pigments and bacteriorhodopsin

A simple model for the early events in visual pigments and bacteriorhodopsin is proposed. The model makes use of the likelihood that a negatively charged amino acid forms a salt bridge with the positively charged nitrogen of the retinylic chromophore. The photochemical event is a cis-trans isomerization in visual pigments and a trans-cis isomerization in bacteriorhodopsin, which in each case cleaves the salt bridge and thus separates charge in the interior of the protein. We propose that this is how the energy of a photon is transduced into chemical free energy of the primary photoproduct. The use of photoisomerization of a flexible chromophore to achieve charge separation provides a general mechanism which may be applicable to other systems. Our model explains many of the fundamental properties of visual pigments and their photoproducts. First, the extraordinarily low rate of thermally populating the ground state of the primary photoproduct, as determined from psychophysical and electrophysiological measurements, is seen as resulting from the large barrier to thermal isomerization about a double bond, perhaps enhanced by electrostatic attraction in the salt bridge. Second, the increase in energy and the spectral red shift that characterize the primary photochemical events are natural consequences of the separation of charge. Proton-dependent processes detected with picosecond techniques are proposed to be ground-state relaxation processes following the primary photochemical event. Finally, the charged groups of the salt bridge, repositioned by photoisomerization, provide a simple mechanism for vectorial proton translocation in bacteriorhodopsin.     

The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling

The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the gamma-phosphate of bound nucleoside 5' triphosphate. In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes. When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased. dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein. gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity. However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA. These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.     

Vibrational energy relaxation in proteins

An overview of theories related to vibrational energy relaxation (VER) in proteins is presented. VER of a selected mode in cytochrome c is studied by using two theoretical approaches. One approach is the equilibrium simulation approach with quantum correction factors, and the other is the reduced model approach, which describes the protein as an ensemble of normal modes interacting through nonlinear coupling elements. Both methods result in similar estimates of the VER time (subpicoseconds) for a CD stretching mode in the protein at room temperature. The theoretical predictions are in accord with previous experimental data. A perspective on directions for the detailed study of time scales and mechanisms of VER in proteins is presented.     

Intramembrane charge movements and excitation- contraction coupling expressed by two-domain fragments of the Ca2+ channel

To investigate the molecular basis of the voltage sensor that triggers excitation-contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (alpha1S 1-670) and the III-IV domain (alpha1S 701-1873) were expressed in dysgenic alpha1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca(2+) transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Q(max) = 3 +/- 0.23 vs. 5.4 +/- 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V(1/2) = 0.2 +/- 3.5 vs. 22 +/- 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal alpha1A Ca(2+) channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the alpha1A I-II domain suggest that this hemi-Ca(2+) channel could be relevant to neuronal function.