Energy Transduction Processes Research Articles

Homeostasis of the membrane proton permeability in Bacillus subtilis grown at different temperatures

Bacillus subtilis was grown at its growth temperature limits and at various temperatures in between the lower and upper growth temperature boundary. Liposomes were made of the extracted membrane lipids derived from these cells. The headgroup composition of the cytoplasmic membrane lipids did not differ significantly at the lower (13°C) and upper (50°C) temperature boundary. The averaged lipid acyl chain length, degree of saturation, and ratio of iso- and anteiso-branched fatty acids increased with the temperature. At the temperature of growth, the membranes were in a liquid–crystalline phase, but liposomes derived from cells grown at 13°C were almost threefold more viscous than those derived from 50°C grown cells. The temperature dependence of the proton permeability of the liposomes was determined using the acid-pulse method with monitoring of the outside pH with the fluorescent probe pyranine. The proton permeability of each liposome preparation increased with the temperature. However, the proton permeability of the liposomes at the growth temperature of the cells from which the lipids were derived was almost constant. These data indicate that the growth temperature dependent variation in lipid acyl chain composition permits maintenance of the proton permeability of the cytoplasmic membrane. This ‘homeo-proton permeability adaptation’ precludes futile cycling of protons at higher growth temperatures and allows cells to sustain the proton motive force as a driving force for essential energy transducing processes.

Time course of reactions controlled and gated by intramolecular dynamics of proteins: predictions of the model of random walk on fractal lattices.

Computer simulations of random walk on the Sierpinski gasket and percolation clusters demonstrate that the short, initial condition-dependent stage of protein involving reactions can dominate the progress of the reaction over the main stage described by the standard kinetics. This phenomenon takes place if the intramolecular conformational transition dynamics modeled by the stochastic process is slow enough and the initial conformational substate of the protein already belongs to the transition state of the reaction. Both conditions are realized in two kinds of experiments: small ligand rebinding to protein after laser flash photolysis and direct recording of single protein channel activity. The model considered suggests simple analytical formulae that can explain the time behavior of the processes observed and its variation with temperature. The initial condition-dependent stage, and not the stage described by the standard kinetics, is expected as responsible for the coupling of component reactions in the complete catalytic cycles and more complex processes of biological free energy transduction.

Computation of the Structure-Dependent pKa Shifts in a Polypentapeptide of the Poly[fv(IPGVG), fE(IPGEG)] Family

We report the results of a theoretical study intended to elucidate the molecular basis of the effect of hydrophobicity on the pKa's of ionizable groups in polypeptides and proteins. In particular, we have focused on a polypentapeptide of a family associated with the process of free energy transduction. Our theoretical calculations, carried out by using a conformational search method in combination with a fast multigrid boundary element method to solve the Poisson equation, led to results that are in good agreement with the experimental observations.

A synthetic picture of intramolecular dynamics of proteins. Towards a contemporary statistical theory of biochemical processes.

An increasing body of experimental evidence indicates the slow character of internal dynamics of native proteins. The important consequence of this is that theories of chemical reactions, used hitherto, appear inadequate for description of most biochemical reactions. Construction of a contemporary, truly advanced statistical theory of biochemical processes will need simple but realistic models of microscopic dynamics of biomolecules. In this review, intended to be a contribution towards this direction, three topics are considered. First, an intentionally simplified picture of dynamics of native proteins which emerges from recent investigations is presented. Fast vibrational modes of motion, of periods varying from 10(-14) to 10(-11) s, are contrasted with purely stochastic conformational transitions. Significant evidence is adduced that the relaxation time spectrum of the latter spreads in the whole range from 10(-11) to 10(5) s or longer, and up to 10(-7) s it is practically quasi-continuous. Next, the essential ideas of the theory of reaction rates based on stochastic models of intramolecular dynamics are outlined. Special attention is paid to reactions involving molecules in the initial conformational substrates confirmed to the transition state, which is realized in actual experimental situations. And finally, the two best experimentally justified classes of models of conformational transition dynamics, symbolically referred to as "protein glass" and "protein machine", are described and applied to the interpretation of a few simple biochemical processes, perhaps the most important result reported is the demonstration of the possibility of predominance of the short initial condition-dependent stage of protein involved reactions over the main stage described by the standard kinetics. This initial stage, and not the latter, is expected to be responsible for the coupling of component reactions in the complete enzymatic cycles as well as more complex processes of biological free energy transduction.

Involvement of aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria

Effects of aspartate, glutamate and an inhibitor of the aspartate/glutamate antiporter, diethylpyrocarbonate (DEPC), on uncoupling of the energy transduction processes in rat liver mitochondria have been investigated. It is found that both the antiporter substrates and the antiporter inhibitor operate as recouplers when uncoupling is caused by free fatty acids (FFA). Recoupling consists in (1) partial inhibition of the FFA-stimulated respiration and (2) some increase in the membrane potential. Half-maximal effects are observed at concentrations of glutamate and aspartate close the K m values of the antiporter. Recouplings by glutamate (aspartate) and DEPC are not additive. On the other hand, recoupling by any of these compounds and carboxyatractylate or ADP appears to be additive. Uncoupling by dinitrophenol is less sensitive to the recouplers whereas that by FCCP is not sensitive at all. It is concluded that uncoupling by FFA in rat liver mitochondria is mediated not only by the ATP/ADP antiporter but also by the aspartate/glutamate antiporter.

The diversity of coenzyme Q function

Coenzyme Q is uniquely designed as an electron and proton carrier within the lipid phase of membranes. It now appears that this unique chemistry has diverse application to important functions in all cellular membranes. The first function of coenzyme Q was defined in the energy transduction process in mitochondria. New studies show that the presence of coenzyme Q in other cellular membranes has dynamic rather than passive significance. Coenzyme Q functions in the plasma membrane electron transport involved in activation of signalling protein kinases related to gene activation for cellular proliferation. Furthermore, the antioxidant potential of the reduced coenzyme Q is now taken on a new significance in the evidence that the reduced quinone can act to maintain tocopherol in the reduced state in membranes and ascorbate reduced both inside and outside the cell.

Phosphate transport in prokaryotes: molecules, mediators and mechanisms.

Bacteria have evolved sophisticated Pi transport systems which combine high affinity with coupling to metabolic energy. This review discusses the current evidence concerning the physiological, biochemical, and molecular properties of these Pi transport systems in prokaryotes. Major developments of the past years will be presented with emphasis on three kinds of issues. First, work on Pi transport in Escherichia coli and the polyphosphate-accumulating Acinetobacter johnsonii has assigned a novel biochemical mechanism and provided additional descriptive information for the transport of Pi and divalent cations. It is therefore appropriate to summarize these new facts and emphasize their general relevance for pro- and eukaryotic cells. Second, recent work on the bioenergetics of Pi transport in A. johnsonii has demonstrated the profound role of the transmembrane Pi gradient in energy transducing processes such as the accumulation of solutes, and the generation of a proton motive force. These findings and their significance for the survival of the cell during metabolic stress conditions will be discussed. Finally, polyphosphate-accumulating microorganisms play a valuable role in biotechnological applications, such as in wastewater treatment. As such organisms are still underrepresented in current molecular microbiological studies, the investigations in A. johnsonii described here may serve as a useful precedent for those to come.

The role of transport processes in survival of lactic acid bacteria. Energy transduction and multidrug resistance.

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural and functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.

Proton gradient-induced changes of the interaction between CF 0 and CF 1 as probed by cleavage with NaSCN

Treatment of isolated thylakoids from spinach chloroplasts with the chaotropic salt NaSCN results in release of CF 1 together with a variety of other proteins. The detachment of the CF 1 polypeptides by 0.5 M NaSCN was much higher in illuminated than in non-illuminated samples. The CF 1 subunits are released at about the same molar ratio as in the entire complex, suggesting that NaSCN cleaves primarily between CF 0 and CF 1. Hoever, subsequent dissociation of CF 1 into smaller aggregates or individual subunits may occur. Detachment of CF 1 from pea thylakoids requires somewhat higher NaSCN concentrations, but a distinct light/dark difference is nevertheless found. The enhancement of CF 1 solubilization by light is largely abolished by the uncoupler nigericin, suggesting that the proton gradient affects the interaction between CF 0 and CF 1. The enhanced release is also reduced by the CF 0 blocker DCCD, indicating that the translocation of protons through CF 0 is necessary. The enhancement is partially abolished by the substrates of phosphorylation, ADP and phosphate, suggesting that the change of interaction between the subcomplexes is connected with the process of energy transduction in CF 0CF 1.

Partial suppression of an Escherichia coli TonB transmembrane domain mutation (ΔV17) by a missense mutation in ExbB

Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The delta V17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17. Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export. Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.

Energy transduction in lactic acid bacteria

In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.

Energy transduction in lactic acid bacteria.

In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.

30] Insertion of lipids and proteins into bacterial membranes by fusion with liposomes

Publisher Summary This chapter discusses the insertion of lipids and proteins into bacterial membranes by fusion with liposomes. Cytoplasmic membrane vesicles derived from Escherichia coli and many other bacteria provide a well-defined model system to study membrane- associated energy-transducing processes. A method has been developed in our laboratory that allows the fusion-mediated insertion of a variety of delta p-generating systems into bacterial membranes while energy conserving properties of the membrane are retained. These procedures also are used for the bulk enrichment of the bacterial membrane with exogenous lipids. Principles of this method and specific details of its applications in studies on the mechanism of solute translocation and lipid protein interactions in lactic acid bacteria are described in this chapter.

Energy transduction and transport processes in thermophilic bacteria.

Bacterial growth at the extremes of temperature has remained a fascinating aspect in the study of membrane function and structure. The stability of the integral membrane proteins of thermophiles make them particularly amenable to study. Respiratory enzymes of thermophiles appear to be functionally similar to the mesophilic enzymes but differ in their thermostability and unusual high turnover rates. Energy coupling at extreme temperatures seems inefficient as suggested by the high maintenance coefficients and the high permeability of the cell membrane to protons. Nevertheless, membranes maintain their structure at these extremes through changes in fatty acid acyl chain composition. Archaebacteria synthesize novel membrane-spanning lipids with unique physical characteristics. Thermophiles have adapted to life at extreme temperatures by using sodium ions rather than protons as coupling ions in solute transport. Genetic and biochemical studies of these systems now reveal fundamental principles of such adaptations. The recent development of reconstitution techniques using membrane-spanning lipids allows a rigorous biochemical characterization of membrane proteins of extreme thermophiles in their natural environment.

Self-organization of polymer-motor systems in the cytoskeleton.

Microtubules and actin filaments are organized into dynamic arrays inside cells. In this paper I discuss in conceptual form the assembly mechanisms of three specific arrays: asters, spindles and leading edge structures. The role of energy transducing processes, particularly motor protein activity, in assembly is explored. I conclude that dynamic interaction between motor proteins and cytoskeletal polymers play a very general role in spatial organization of the cytoplasm.

Cytochrome c oxidase: the presumptive channel holds at least four water molecules

The catalytic cycle of cytochrome oxidase requires that different intermediates be differentially hydrated. The movement of water, into and out of the enzyme, is essential for but is not directly linked to electron transfer. For this reason it is felt that the water cycle represents an aspect of the energy transduction process. We show that a minimum of four waters can be trapped in the oxidase during arrested turnover.

Cytochrome c oxidase: the presumptive channel holds at least four water molecules

The catalytic cycle of cytochrome oxidase requires that different intermediates be differentially hydrated. The movement of water, into and out of the enzyme, is essential for but is not directly linked to electron transfer. For this reason it is felt that the water cycle represents an aspect of the energy transduction process. We show that a minimum of four waters can be trapped in the oxidase during arrested turnover.

Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans

Formate-grown cells of the obligately chemolithoautotrophic acidophile Thiobacillus ferrooxidans were capable of formate- and elemental sulfur-dependent reduction of ferric iron under anaerobic conditions. Under aerobic conditions, both oxygen and ferric iron could be simultaneously used as electron acceptors. To investigate whether anaerobic ferric iron respiration by T. ferrooxidans is an energy-transducing process, uptake of amino acids was studied. Glycine uptake by starved cells did not occur in the absence of an electron donor, neither under aerobic conditions nor under anaerobic conditions. Uptake of glycine could be driven by formate- and ferrous iron-dependent oxygen uptake. Under anaerobic conditions, ferric iron respiration with the electron donors formate and elemental sulfur could energize glycine uptake. Glycine uptake was inhibited by the uncoupler 2,4-dinitrophenol. The results indicate that anaerobic ferric iron respiration can contribute to the energy budget of T. ferrooxidans.

Paracoccus denitrificans cytochrome c1 gene replacement mutants

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.

Light Harvesting Molecular Assemblies in the Design of Highly Luminescent Sol-Gel Derived Glasses

ABSTRACTSol-gel derived molecular composites exhibiting intense luminescence, induced from efficient energy transduction processes, have been prepared. The composites are comprised of an Eu3+⊂ 2.2.1 cryptate complex or native Eu3+ ion embedded in sol-gel derived titania glass films. The titania glasses contain interconnected porous networks that permit the diffusion of exogenous substrates, such as the salts of benzoic and 4-tert-butylbenzoic acids, through the film. Interaction of the substrate with the embedded lanthanide complex is indicated by enhanced luminescence from the lanthanide ion. The carboxylic acid salts whose electronic excited states are produced upon capture of incident photons, undergo facile transfer of their electronic energy to the lanthanide ion. By monitoring europium ion luminescence, the diffusion constants of the benzoate and 4-tert-butylbenzoate salts have been measured. Although the diffusion of the 4-tert-butylbenzoate is slower than that of benzoate, the overall higher sensitivity of the former is consistent with hydrophobic guest-host interactions. These new molecular composites relying on the immobilization of an absorption-energytransfer- emission molecular assembly in porous, optically transparent ceramic glasses may be useful in the design of practical sensing devices.