Proton Electrochemical Potential Research Articles

Low Dielectric Permittivity of Water at the Membrane Interface: Effect on the Energy Coupling Mechanism in Biological Membranes

Protonmotive force (the transmembrane difference in electrochemical potential of protons, Δ μ ˜ H + ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of Δ μ ˜ H + relates to the difference in the proton activity between two bulk water phases (ΔpH B) or between two membrane surfaces (ΔpH S). To scrutinize whether ΔpH S can deviate from ΔpH B, we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, Phys. Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5–1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2–0.3 eV) than for monovalent cations (0.1–0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton “pumps” at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH S of ∼6.0; the value of pH S was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.

Changed Energy State and Increased Mitochondrial β-Oxidation Rate in Liver of Rats Associated with Lowered Proton Electrochemical Potential and Stimulated Uncoupling Protein 2 (UCP-2) Expression: EVIDENCE FOR PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-α INDEPENDENT INDUCTION OF UCP-2 EXPRESSION

Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) alpha-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, beta-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Delta(p)) was 15% lower than controls. The change affected the Delta(Psi) component only, leaving the (Delta)pH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARalpha-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARdelta, we discuss how this subtype might compensate for deficiency of PPARalpha. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others.

Amino acid transport by Sphingomonas sp. strain Ant 17 isolated from oil-contaminated Antarctic soil

The Antarctic bacterial isolate Sphingomonas sp. strain Ant 17 utilized a wide range of L-isomer amino acids as the sole carbon and energy source for growth. The pH and temperature optima for growth on amino acids were pH 7.0 and 15°C, respectively. Growth on serine and tryptophan was inhibited by uncouplers and inhibitors of oxidative phosphorylation, but not by monensin, a Na+/H+ antiporter, suggesting that sodium gradients were not specifically required for growth on these amino acids. Serine transport was via a high-affinity (apparent Km of 8 μM) permease specific for both the L- and D-isomer. Tryptophan transport exhibited biphasic kinetics with both high-affinity (apparent Km of 2.5 μM) and low-affinity (non-saturable) uptake systems detected. The high-affinity system was specific for L-tryptophan, L-tyrosine, and L-phenylalanine whereas the low-affinity permease was specific for L-tryptophan and L-phenylalanine, but not L-tyrosine. Neither orthovanadate nor sodium arsenate, inhibitors of ATP-dependent permeases, had any significant inhibitory effect on the rate of serine and tryptophan transport. The protonophore carbonyl cyanide m-chlorophenylhydrazone completely abolished serine and tryptophan transport; maximum rates of solute uptake were observed at acidic pH values (pH 4.0–5.0) for both amino acids. These results suggest that an electrochemical potential of protons is the driving force for serine and tryptophan transport by Ant 17. These high-affinity proton-driven permeases function over environmental extremes (e.g. broad temperature and pH range) that are likely to prevail in the natural habitat of this bacterium.

The role of electrostatics in proton-conducting membrane protein complexes

Electrostatic interactions play a key role in the coupling of electron and proton transfer in membrane protein complexes during the conversion of the energy stored in sunlight or reduced substrates into biochemical energy via a transmembrane electrochemical proton potential. Principles of charge stabilization within membrane proteins are reviewed and discussed for photosynthetic reaction centers, cytochrome c oxidases, and diheme-containing quinol:fumarate reductases. The impact of X-ray structure-based electrostatic calculations on the functional interpretation of these structural coordinates, on providing new explanations for experimental observations, and for the design of more focused additional experiments is illustrated by a number of key examples.

A mutation in subunit I of cytochrome oxidase from Rhodobacter sphaeroides results in an increase in steady-state activity but completely eliminates proton pumping.

The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.

Enzymology and bioenergetics of respiratory nitrite ammonification.

Nitrite is widely used by bacteria as an electron acceptor under anaerobic conditions. In respiratory nitrite ammonification an electrochemical proton potential across the membrane is generated by electron transport from a non-fermentable substrate like formate or H(2) to nitrite. The corresponding electron transport chain minimally comprises formate dehydrogenase or hydrogenase, a respiratory quinone and cytochrome c nitrite reductase. The catalytic subunit of the latter enzyme (NrfA) catalyzes nitrite reduction to ammonia without liberating intermediate products. This review focuses on recent progress that has been made in understanding the enzymology and bioenergetics of respiratory nitrite ammonification. High-resolution structures of NrfA proteins from different bacteria have been determined, and many nrf operons sequenced, leading to the prediction of electron transfer pathways from the quinone pool to NrfA. Furthermore, the coupled electron transport chain from formate to nitrite of Wolinella succinogenes has been reconstituted by incorporating the purified enzymes into liposomes. The NrfH protein of W. succinogenes, a tetraheme c-type cytochrome of the NapC/NirT family, forms a stable complex with NrfA in the membrane and serves in passing electrons from menaquinol to NrfA. Proteins similar to NrfH are predicted by open reading frames of several bacterial nrf gene clusters. In gamma-proteobacteria, however, NrfH is thought to be replaced by the nrfBCD gene products. The active site heme c group of NrfA proteins from different bacteria is covalently bound via the cysteine residues of a unique CXXCK motif. The lysine residue of this motif serves as an axial ligand to the heme iron thus replacing the conventional histidine residue. The attachment of the lysine-ligated heme group requires specialized proteins in W. succinogenes and Escherichia coli that are encoded by accessory nrf genes. The proteins predicted by these genes are unrelated in the two bacteria but similar to proteins of the respective conventional cytochrome c biogenesis systems.

2-Oxoglutarate transport system in S taphylococcus aureus

2-[(14)C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H(+) symporter (K(m)=0.105 mM), energized by an electrochemical proton potential (Delta mu H(+)) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a Delta mu H(+)-independent facilitated diffusion system (K(m)=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H(+) symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by Delta mu H(+) generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates Delta mu H(+) for further, autoenergized transport of this 2-oxoacid and also for Delta mu H(+)-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd(2+) accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of Delta mu H(+) generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd(2+) does not affect energy conservation due to its extrusion by the Cd(2+) efflux system (Cd(2+)-ATPase of P-type), which prevents Cd(2+) accumulation.

Conserved Residues Ser16 and His20 and Their Relative Positioning Are Essential for TonB Activity, Cross-linking of TonB with ExbB, and the Ability of TonB to Respond to Proton Motive Force

The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31). Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser(16) to His(20) was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp(11) resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp(11) was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.

Transport of C(4)-dicarboxylates in Wolinella succinogenes.

C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.

A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes.

Wolinella succinogenes can grow by anaerobic respiration with nitrate or nitrite using formate as electron donor. Two forms of nitrite reductase were isolated from the membrane fraction of W. succinogenes. One form consisted of a 58 kDa polypeptide (NrfA) that was identical to the periplasmic nitrite reductase. The other form consisted of NrfA and a 22 kDa polypeptide (NrfH). Both forms catalysed nitrite reduction by reduced benzyl viologen, but only the dimeric form catalysed nitrite reduction by dimethylnaphthoquinol. Liposomes containing heterodimeric nitrite reductase, formate dehydrogenase and menaquinone catalysed the electron transport from formate to nitrite; this was coupled to the generation of an electrochemical proton potential (positive outside) across the liposomal membrane. It is concluded that the electron transfer from menaquinol to the catalytic subunit (NrfA) of W. succinogenes nitrite reductase is mediated by NrfH. The structural genes nrfA and nrfH were identified in an apparent operon (nrfHAIJ) with two additional genes. The gene nrfA encodes the precursor of NrfA carrying an N-terminal signal peptide (22 residues). NrfA (485 residues) is predicted to be a hydrophilic protein that is similar to the NrfA proteins of Sulfurospirillum deleyianum and of Escherichia coli. NrfH (177 residues) is predicted to be a membrane-bound tetrahaem cytochrome c belonging to the NapC/NirT family. The products of nrfI and nrfJ resemble proteins involved in cytochrome c biogenesis. The C-terminal third of NrfI (902 amino acid residues) is similar to CcsA proteins from Gram-positive bacteria, cyanobacteria and chloroplasts. The residual N-terminal part of NrfI resembles Ccs1 proteins. The deduced NrfJ protein resembles the thioredoxin-like proteins (ResA) of Helicobacter pylori and of Bacillus subtilis, but lacks the common motif CxxC of ResA. The properties of three deletion mutants of W. succinogenes (DeltanrfJ, DeltanrfIJ and DeltanrfAIJ) were studied. Mutants DeltanrfAIJ and DeltanrfIJ did not grow with nitrite as terminal electron acceptor or with nitrate in the absence of NH4+ and lacked nitrite reductase activity, whereas mutant DeltanrfJ showed wild-type properties. The NrfA protein formed by mutant DeltanrfIJ seemed to lack part of the haem C, suggesting that NrfI is involved in NrfA maturation.

The Proteolipid of the A1A0ATP Synthase from Methanococcus jannaschii Has Six Predicted Transmembrane Helices but Only Two Proton-translocating Carboxyl Groups

The proteolipid, a hydrophobic ATPase subunit essential for ion translocation, was purified from membranes of Methanococcus jannaschii by chloroform/methanol extraction and gel chromatography and was studied using molecular and biochemical techniques. Its apparent molecular mass as determined in SDS-polyacrylamide gel electrophoresis varied considerably with the conditions applied. The N-terminal sequence analysis made it possible to define the open reading frame and revealed that the gene is a triplication of the gene present in bacteria. In some of the proteolipids, the N-terminal methionine is excised. Consequently, two forms with molecular masses of 21,316 and 21,183 Da were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The molecular and biochemical data gave clear evidence that the mature proteolipid from M. jannaschii is a triplication of the 8-kDa proteolipid present in bacterial F(1)F(0) ATPases and most archaeal A(1)A(0) ATPases. Moreover, the triplicated form lacks a proton-translocating carboxyl group in the first of three pairs of transmembrane helices. This finding puts in question the current view of the evolution of H(+) ATPases and has important mechanistic consequences for the structure and function of H(+) ATPases in general.

Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase

The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the γ-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the γ-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 γ-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.

Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase

The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the gamma-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the gamma-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 gamma-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.

Properties of uracil transport by vegetative mycelium of Trichoderma viride

The transport of radioactively labelled uracil into submerged mycelium of T. viride was measured by means of a membrane filtration technique. It was found to be time-dependent (up to 90 min) and concentration-dependent (up to 8 mmol l −1). Its concentration dependence was biphasic and consisted from the saturatable part (at the uracil concentration below 0.2 mmol l −1) with K M=0.08±0.02 mmol l −1 and V max=1.74±0.3 nmol (mg dry wt.) −1 h −1, and from the region at higher uracil concentration which showed only a weak saturatability with the substrate. The transport measured in the saturatable part of the curve was also pH- and temperature-dependent. The optimal pH was between 5.4 and 6.4 and the optimal temperature was at 37°C. The activation energy of 54 kJ mol −1 and the temperature quotient of Q 10=2.1 could be calculated from the temperature dependence. The entry of uracil was in part inhibited by nucleobases and their analogues, nucleosides, nucleotides and amino acids. The inhibitors had similar inhibitory efficiency about 50% at 0.2 mmol l −1. 3,3′,4′,5-tetrachlorosalicylanilide (TCS), the uncoupling agent, significantly inhibited the uracil transport, but its inhibitory efficiency decreased upon increasing the uracil concentration. Ionophore antibiotics valinomycin and monensin also inhibited the uracil transport. Inhibitors of RNA-polymerase, rifamycin and rifampicin were without effect. The results suggest that at low uracil concentrations (below 0.2 mmol l −1), its transport is mediated by a carrier and is driven by the electrochemical potential of protons. At higher uracil concentrations, the transport may be driven by the concentration difference of uracil with the contribution of the proton-motive force. It is feasible that inhibitors of uracil transport tested exert their inhibition by the dissipation of the driving force rather than by the direct competition with the substrate-binding site.

Properties of uracil transport by vegetative mycelium of Trichoderma viride.

The transport of radioactively labelled uracil into submerged mycelium of T. viride was measured by means of a membrane filtration technique. It was found to be time-dependent (up to 90 min) and concentration-dependent (up to 8 mmol l-1). Its concentration dependence was biphasic and consisted from the saturatable part (at the uracil concentration below 0.2 mmol l-1) with KM = 0.08 +/- 0.02 mmol l-1 and Vmax = 1.74 +/- 0.3 nmol (mg dry wt.)-1 h-1, and from the region at higher uracil concentration which showed only a weak saturatability with the substrate. The transport measured in the saturatable part of the curve was also pH- and temperature-dependent. The optimal pH was between 5.4 and 6.4 and the optimal temperature was at 37 degrees C. The activation energy of 54 kJ mol-1 and the temperature quotient of Q10 = 2.1 could be calculated from the temperature dependence. The entry of uracil was in part inhibited by nucleobases and their analogues, nucleosides, nucleotides and amino acids. The inhibitors had similar inhibitory efficiency about 50% at 0.2 mmol l-1. 3,3',4',5-tetrachlorosalicylanilide (TCS), the uncoupling agent, significantly inhibited the uracil transport, but its inhibitory efficiency decreased upon increasing the uracil concentration. Ionophore antibiotics valinomycin and monensin also inhibited the uracil transport. Inhibitors of RNA-polymerase, rifamycin and rifampicin were without effect. The results suggest that at low uracil concentrations (below 0.2 mmol l-1), its transport is mediated by a carrier and is driven by the electrochemical potential of protons. At higher uracil concentrations, the transport may be driven by the concentration difference of uracil with the contribution of the protonmotive force. It is feasible that inhibitors of uracil transport tested exert their inhibition by the dissipation of the driving force rather than by the direct competition with the substrate-binding site.

An electrostatic model of the bacterial flagellar motor

The rotation of bacterial flagella is driven by the difference in electrochemical potential for protons between the periplasmic and the cytoplasmic space. This requires a coupling between a flow of protons across the cell membrane and the rotation of a macromolecular complex representing the rotor of the bacterial flagellar motor. The mechanism of coupling is not as yet understood. A model for the motor is described based on electrostatic interaction between fixed charges on the circumference of the rotor and protons moving through channels in the membrane. The design of the model takes into account the presently known structural and functional aspects of the motor. It can successfully simulate a large number of diverse experimental data with one appropriately adjusted set of parameter values. This also includes data which other models fail to account for quantitatively, such as the stoichiometry of protons per revolution and the dependence of the swimming speed of cells on the pH in the suspending medium.

Isolation and Characterization of the Gene (CLS1 ) Encoding Cardiolipin Synthase in Saccharomyces cerevisiae

In eukaryotic cells, cardiolipin (CL) synthase catalyzes the final step in the synthesis of CL from phosphatidylglycerol and CDP-diacylglycerol. CL and its synthesis are localized predominantly to the mitochondrial inner membrane, and CL is generally thought to be an essential component of many mitochondrial processes. By using homology searches for genes potentially encoding phospholipid biosynthetic enzymes, we have cloned the gene (CLS1) encoding CL synthase in Saccharomyces cerevisiae. Overexpression of the CLS1 gene under its endogenous promoter or the inducible GAL1 promoter in yeast and expression of CLS1 in baculovirus-infected insect cells resulted in elevated CL synthase activity. Disruption of the CLS1 gene in a haploid yeast strain resulted in the loss of CL synthase activity, no detectable CL, a 5-fold elevation in phosphatidylglycerol levels, and lack of staining of mitochondria by a dye with high affinity for CL. The cls1::TRP1 null mutant grew on both fermentable and non-fermentable carbon sources but more poorly on the latter. The level and activity of cytochrome c oxidase was normal, and a dye whose accumulation is dependent on membrane proton electrochemical potential effectively stained the mitochondria. These results definitively identify the gene encoding the CL synthase of yeast.

Inhibitor Binding within the NarI Subunit (Cytochromebnr) of Escherichia coli Nitrate Reductase A

We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochrome bnr (NarI) of Escherichia coli nitrate reductase (NarGHI). Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity with I50 values of 0.25 and 6 microM, respectively, and prevent the generation of a NarGHI-dependent proton electrochemical potential across the cytoplasmic membrane. These inhibitors have little effect on the rate of reduction of the two hemes of NarI (bL and bH), but have an inhibitory effect on the extent of nitrate-dependent heme reoxidation. No quinol-dependent heme bH reduction is detected in a mutant lacking heme bL (NarI-H66Y), whereas a slow but complete heme bL reduction is detected in a mutant lacking heme bH (NarI-H56R). This is consistent with physiological quinol binding and oxidation occurring at a site (QP) associated with heme bL which is located toward the periplasmic side of NarI. Optical and EPR spectroscopies performed in the presence of stigmatellin or HOQNO provide further evidence that these inhibitors bind at a heme bL-associated QP site. These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of heme bL and electron transfer through heme bH to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.

Light-driven production of ATP catalysed by F0F1-ATP synthase in an artificial photosynthetic membrane.

Energy-transducing membranes of living organisms couple spontaneous to non-spontaneous processes through the intermediacy of protonmotive force (p.m.f.)--an imbalance in electrochemical potential of protons across the membrane. In most organisms, p.m.f. is generated by redox reactions that are either photochemically driven, such as those in photosynthetic reaction centres, or intrinsically spontaneous, such as those of oxidative phosphorylation in mitochondria. Transmembrane proteins (such as the cytochromes and complexes I, III and IV in the electron-transport chain in the inner mitochondrial membrane) couple the redox reactions to proton translocation, thereby conserving a fraction of the redox chemical potential as p.m.f. Many transducer proteins couple p.m.f. to the performance of biochemical work, such as biochemical synthesis and mechanical and transport processes. Recently, an artificial photosynthetic membrane was reported in which a photocyclic process was used to transport protons across a liposomal membrane, resulting in acidification of the liposome's internal volume. If significant p.m.f. is generated in this system, then incorporating an appropriate transducer into the liposomal bilayer should make it possible to drive a non-spontaneous chemical process. Here we report the incorporation of F0F1-ATP synthase into liposomes containing the components of the proton-pumping photocycle. Irradiation of this artificial membrane with visible light results in the uncoupler- and inhibitor-sensitive synthesis of adenosine triphosphate (ATP) against an ATP chemical potential of approximately 12 kcal mol(-1), with a quantum yield of more than 7%. This system mimics the process by which photosynthetic bacteria convert light energy into ATP chemical potential.

PHYSIOLOGY OF ION TRANSPORT ACROSS THE TONOPLAST OF HIGHER PLANTS.

The vacuole of plant cells plays an important role in the homeostasis of the cell. It is involved in the regulation of cytoplasmic pH, sequestration of toxic ions and xenobiotics, regulation of cell turgor, storage of amino acids, sugars and CO2 in the form of malate, and possibly as a source for elevating cytoplasmic calcium. All these activities are driven by two primary active transport mechanisms present in the vacuolar membrane (tonoplast). These two mechanisms employ high-energy metabolites to pump protons into the vacuole, establishing a proton electrochemical potential that mediates the transport of a diverse range of solutes. Within the past few years, great advances at the molecular and functional levels have been made on the characterization and identification of these mechanisms. The aim of this review is to summarize these studies in the context of the physiology of the plant cell.