Non-physiological Electron Donor Research Articles

Characterization of a cytoplasmic 2-Cys peroxiredoxin from Citrus sinensis and its potential role in protection from oxidative damage and wound healing

In present work, the recombinant cytoplasmic 2-Cys peroxiredoxin from Citrus sinensis (CsPrx) was purified and characterized. The peroxidase activity was examined with different substrates using DTT, a non-physiological electron donor. The conformational studies, in oxidized and reduced states, were performed using circular dichroism (CD) and fluorescence measurement. The CD analysis showed higher α-helical content for reduced state of the protein. The thermal stability studies of CsPrx by Differential Scanning Calorimetry (DSC) showed that oxidized state is more stable as compared to the reduced state of CsPrx. In vitro studies showed that the CsPrx provides a protective shield against ROS and free radicals that participate in the degradation of plasmid DNA. The pre-treatment of 10 μM CsPrx provide almost 100% protection against peroxide-mediated cell killing in the Vero cells. CsPrx showed significant cell proliferation and wound healing properties. The superior morphology of viable cells and wound closure was found at 20 μM CsPrx treated for 12 h. The results demonstrated that CsPrx is a multifaceted protein with a significant role in cell proliferation, wound healing and protection against hydrogen peroxide-induced cellular damage. This could be the first report of a plant peroxiredoxin being characterized for biomedical applications.

In vitro Nitrate Reductase Activity Assay of Mycolicibacterium smegmatis Crude Extract.

Nitrate is one of the major inorganic nitrogen sources for microorganisms. Many bacterial and archaeal lineages can express cytoplasmic assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite in the nitrate assimilation pathway. Here, we present a detailed protocol for measuring in vitro nitrate reductase (NaR) activity of NAS enzymes from Mycolicibacterium smegmatis crude extract using both physiological and non-physiological electron donors.

A novel approach to measure mitochondrial respiration in frozen biological samples.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Characterization of a bacterioferritin comigratory protein family 1-Cys peroxiredoxin from Candidatus Liberibacter asiaticus.

To defend against the lethality of the reactive oxygen species (ROS), nature has armed microorganisms with a range of antioxidant proteins. These include peroxiredoxin (Prx) super family proteins which are ubiquitous cysteine-based non-heme peroxidases. The phytopathogenic bacterium Candidatus Liberibacter asiaticus (CLA), an etiological agent of citrus plants diseases, posses many genes for defense against oxidative stress. The bacterioferritin comigratory protein (BCP), a member of Prxs, is part of an oxidative stress defense system of CLA. The key residue of these enzymes is peroxidatic Cys (termed CPSH) which is contained within an absolutely conserved PXXX (T/S) XXC motif. In the present study, a 1-Cys Prx enzyme (CLa-BCP), having CPSH/sulfenic acid cysteine (C-46) but lacking the resolving cysteine (CRSH), was characterized from CLA. The peroxidase activity was demonstrated using a non-physiological electron donor DTT against varied substrates. The protein was shown to have the defensive role against peroxide-mediated cell killing and an antioxidant activity. In vitro DNA-binding studies showed that this protein can protect supercoiled DNA from oxidative damage. To the best of our knowledge, this is the first report on a 1-Cys BCPs to have an intracellular reactive oxygen species scavenging activity.

Evidence That the Pi Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle.

Nitrogenase reduction of dinitrogen (N2) to ammonia (NH3) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, Pi release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein-protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the Pi release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle.

Identification of Amino Acids at the Catalytic Site of a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase.

An in silico model of the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942, and information about active sites in related enzymes, had identified Cys148, Met149, Met306, Asp163, and Arg351 as amino acids likely to be involved in either nitrate binding, prosthetic group binding, or catalysis. Site-directed mutagenesis was used to alter each of these residues, and differences in enzyme activity and substrate binding of the purified variants were analyzed. In addition, the effects of these replacements on the assembly and properties of the Mo cofactor and [4Fe-4S] centers were investigated using Mo and Fe determinations, coupled with electron paramagnetic resonance spectroscopy. The C148A, M149A, M306A, D163N, and R351Q variants were all inactive with either the physiological electron donor, reduced ferredoxin, or the nonphysiological electron donor, reduced methyl viologen, as the source of electrons, and all exhibited changes in the properties of the Mo cofactor. Charge-conserving D163E and R351K variants were also inactive, suggesting that specific amino acids are required at these two positions. The implications for the role of these five conserved active-site residues in light of these new results and previous structural, spectroscopic, and mutagenesis studies for related periplasmic nitrate reductases are discussed.

Ligand-Binding and Substrate Turnover of a Heme Peroxidase from the Diatom Thalassiosira Pseudonana

Iron is sparingly soluble in water; therefore, iron-containing enzymes in marine organisms such as diatoms must have critical physiological roles to justify the use of such a precious resource. We have identified a putative heme peroxidase of unknown function, Tp 21683, from the published genome of the model diatom Thalassiosira pseudonana. Conserved domain analysis suggests Tp 21683 consists of an N-terminal kringle domain, likely involved in ligand binding and localization, and a C-terminal b-type heme peroxidase domain. To dissect the function of the two domains of Tp 21683, we have optimized recombinant expression in Escherichia coli and subsequent refolding and purification of the isolated kringle and peroxidase domains. We report preliminary studies to identify the ligand-binding and peroxidase turnover properties of Tp 21683 toward a better understanding of the physiological role of peroxidases in diatoms. We show by intrinsic tryptophan fluorescence that unlike canonical kringle domains from human plasminogen, the kringle domain of Tp 21683 does not show measurable binding to lysine. However, the kringle domain appears to bind to the oleic acid with a Kd of approximately 1 μM. The peroxidase domain is able to turn over hydrogen peroxide using both the nonphysiological electron donor ABTS and the aromatic compound guaiacol with kcat of approximately 40 min-1 and 4 min-1, respectively. These data are the first analysis of a recombinant peroxidase from a diatom, and provide a basis for further investigation of the range of possible ligands and chemical reactions catalyzed by Tp 21683.

Roles of Four Conserved Basic Amino Acids in a Ferredoxin-Dependent Cyanobacterial Nitrate Reductase

The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.

Molecular Basis for Directional Electron Transfer

Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 A, the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.

Chemical modification studies of tryptophan, arginine and lysine residues in maize chloroplast ferredoxin:sulfite oxidoreductase

The ferredoxin-dependent sulfite reductase from maize was treated, in separate experiments, with three different covalent modifiers of specific amino acid side chains. Treatment with the tryptophan-modifying reagent, N-bromosuccinimide (NBS), resulted in a loss of enzymatic activity with both the physiological donor for the enzyme, reduced ferredoxin, and with reduced methyl viologen, a non-physiological electron donor. Formation of the 1:1 ferredoxin/sulfite reductase complex prior to treating the enzyme with NBS completely protected the enzyme against the loss of both activities. Neither the secondary structure, nor the oxidation-reduction midpoint potential (Em) values of the siroheme and [4Fe-4S] cluster prosthetic groups of sulfite reductase, nor the binding affinity of the enzyme for ferredoxin were affected by NBS treatment. Treatment of sulfite reductase with the lysine-modifying reagent, N-acetylsuccinimide, inhibited the ferredoxin-linked activity of the enzyme without inhibiting the methyl viologen-linked activity. Complex formation with ferredoxin protects the enzyme against the inhibition of ferredoxin-linked activity produced by treatment with N-acetylsuccinimide. Treatment of sulfite reductase with N-acetylsuccinimide also decreased the binding affinity of the enzyme for ferredoxin. Treatment of sulfite reductase with the arginine-modifying reagent, phenylglyoxal, inhibited both the ferredoxin-linked and methyl viologen-linked activities of the enzyme but had a significantly greater effect on the ferredoxin-dependent activity than on the reduced methyl viologen-linked activity. The effects of these three inhibitory treatments are consistent with a possible role for a tryptophan residue the catalytic mechanism of sulfite reductase and for lysine and arginine residues at the ferredoxin-binding site of the enzyme.

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding: THE STRUCTURAL CHANGES TRIGGER ELECTRON TRANSFER TO OXY-P450CAM FROM ELECTRON DONORS

To investigate the functional and structural characterization of a crucial cytochrome P450cam (P450cam)-putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The 1H NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu-358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from d-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and down-field, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for d-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507-14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836-42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.

Complex formation between ferredoxin and Synechococcus ferredoxin:nitrate oxidoreductase

The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.

Identification of an assimilatory nitrate reductase in mutants of Paracoccus denitrificans GB17 deficient in nitrate respiration

A Paracoccus denitrificans strain (M6Omega) unable to use nitrate as a terminal electron acceptor was constructed by insertional inactivation of the periplasmic and membrane-bound nitrate reductases. The mutant strain was able to grow aerobically with nitrate as the sole nitrogen source. It also grew anaerobically with nitrate as sole nitrogen source when nitrous oxide was provided as a respiratory electron acceptor. These growth characteristics are attributed to the presence of a third, assimilatory nitrate reductase. Nitrate reductase activity was detectable in intact cells and soluble fractions using nonphysiological electron donors. The enzyme activity was not detectable when ammonium was included in the growth medium. The results provide an unequivocal demonstration that P. denitrificans can express an assimilatory nitrate reductase in addition to the well-characterised periplasmic and membrane-bound nitrate reductases.

Energy-dependence of the Assimilatory Nitrate Uptake in Azorhizobium caulinodans Strain IRBG 46

Nitrate assimilation by suspensions of Azorhizobium caulinodans strain IRBG 46, as determined by disappearance of nitrate ions from the external medium, displayed the requirement of readily utilizable carbon source. Nitrate uptake was blocked by the uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone and by an inhibitor of ATPase, N, N — dicyclohexyl carbodiimide. The inhibition of nitrate assimilation in the absence of appropriate carbon source was not overcome by the non-physiological terminal electron donor ascorbate plus N-methyl phenazinium methyl sulphate, a substrate combination that allows electron transfer to O2 without the synthesis of ATP. These data suggest that transport of nitrate into the cell is directly dependent on ATP.

Nitrite causes reversible inactivation of nitrate reductase in the yeast Hansenula anomala.

The addition of nitrite, the product of the reaction catalysed by nitrate reductase, to cell suspensions of the yeast Hansenula anomala caused a reversible inactivation of NADPH-dependent nitrate reductase activity. The haem- and Mo-dependent and Mo-dependent activities of nitrate reductase, determined with the non-physiological electron donors FMNH2 and reduced methyl viologen respectively, were less affected. A similar inactivation was found with the proton ionophores 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The inactive enzyme was found in the particulate fraction and cosedimented with the mitochondrial fraction. When the NADPH-dependent nitrate reductase activity was restored in vivo the enzyme was found in the soluble fraction. The inactivation of nitrate reductase by nitrite, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone was dependent on the external pH. The treatment of isolated mitochondria at alkaline pH with Triton X-100 solubilized about 30% of the inactive enzyme.

The effect of lysine- and arginine-modifying reagents on spinach ferredoxin: nitrite oxidoreductase

Treatment of ferredoxin-dependent nitrite reductase, isolated from spinach leaves, with either the lysine-modifying reagent N-acetyl succinimide or the arginine-modifying reagent phenylglyoxal, caused substantial loss of enzymatic activity when reduced ferredoxin, the physiological donor, served as the source of electrons for nitrite reduction. In contrast, treatment with either modifier had little effect on the ability of the enzyme to catalyze nitrite reduction when a non-physiological electron donor, reduced methyl viologen, was supplied. Ferredoxin, but not nitrite, protected the enzyme against loss of ferredoxin-dependent activity caused by both modifiers. Modification caused only minor changes in the absorbance and circular dichroism spectra of nitrite reductase, suggesting that neither N-acetyl succinimide nor phenylglyoxal treatment causes a major conformational change in the enzyme. Modification of nitrite reductase with either N-acetyl succinimide or phenylglyoxal caused the enzyme to lose the ability to form a tight, electrostatically-stabilized complex with ferredoxin. These results have been interpreted in terms of the likely presence of both lysine- and arginine-residues at the ferredoxin-binding domain of spinach nitrite reductase.

Characterization and application of a thermostable primary transport system: Cytochrome‐C oxidase from Bacillus stearothermophilus

Cytochrome-c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE-cellulose, hydroxyapatite- and gel-filtration chromatography. The enzyme is a typical cytochrome-aa3-type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide. The purified enzyme is composed of three different subunits (57, 37 and 22 kDa). The subunit with intermediate molecular mass contains a covalently attached heme-c moiety. The enzyme appeared to be extremely thermostable (inactivation temperature = 81 degrees C). Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non-physiological electron donors like N,N,N',N'-tetramethyl-p-phenylenediamine and phenazine methosulphate. The reconstituted enzyme can generate a proton-motive force consisting of a high membrane potential and trans-membrane pH gradient. The high electro-motive force of the enzyme (delta p = -180 to -200 mV) indicates that this enzyme functions as a high-capacity electrogenic proton pump. Liposomes containing the purified thermostable and thermoactive cytochrome-c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicum. In the hybrid system a high proton-motive force can be generated upon oxidation of reduced N,N,N',N'-tetramethyl-p-phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids. This demonstrates the applicability of the cytochrome-c oxidase to study solute transport in membranes of fermentative bacteria.

Interaction of ferredoxin-linked nitrite reductase with ferredoxin

The native, ferredoxin-linked, form ( M r = 85 000) of nitrite reductase (ferredoxin:nitrite oxidoreductase, EC 1.7.7.1) forms a complex with ferredoxin that can be detected either by enzyme binding to a ferredoxin-Sepharose 4B affinity column or by spectral changes produced when the two proteins are mixed. The K d for ferredoxin binding, estimated to be 6.3·10 −7 M, was increased to 1.1·10 −5 M in the presence of nitrite, indicating lower affinity for ferredoxin binding in the ternary nitrite-ferredoxin-enzyme complex. The native enzyme could be split into a modified form ( M 4 = 61 000) that retains activity with the non-physiological electron donor, methyl viologen, but has lost most of the native enzyme's ferredoxin-linked activity and an M r = 24 000 fragment (coupling protein). The coupling protein, but not the modified nitrite reductase, binds to a ferredoxin-Sepharose 4B affinity column. These results are discussed in terms of a ferredoxin-binding domain located on the coupling protein portion of the native enzyme.

Reconstitution of reduced nicotinamide adenine dinucleotide oxidase activity with menadione in membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D. Relation between electron transfer and active transport.

Membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D contain a low content of menaquinone and consequently oxidaze reduced nicotinamide adenine dinucleotide (NADH) at low rate. Supplementation of the membrane vesicles suspension with the menaquinone-analogue menadione, results in an incorporation of menadione in the membranes. The incorporated menadione increases with the external menadione concentration up to a maximum of 7 nmol of menadione bound per mg membrane protein. The NADH oxidase activity of the membrane vesicles increases linearly with the menadione content and a 35-fold stimulation is obtained in fully reconstituted membrane vesicles; this maximal NADH oxidase activity is about two-fold higher than the NADH oxidase activity in membrane vesicles from wild-type B.subtilis W23. Supplementation of membrane vesicles from B.subtilis W23 with menadione also results in a stimulation of the NADH oxidase activity but only a stimulation of 1.6-fold is maximally obtained. The NADH oxidase activities in reconstituted B.subtilis aro D and B.subtilis W23 membrane vesicles are similarly affected by respiratory chain inhibitors, indicating that menadione occupies physiological sites of menaquinone. NADH and the non-physiological electron donor ascorbate + phenazine methosulphate are the best energy sources for active amino acid transport in membrane vesicles from B.subtilis W23. Membrane vesicles from B.subtilis aro D accumulate amino acids in the presence of acorbate + phenazine methosulphate, but not with NADH. However, membrane vesicles from this mutant, reconstituted with menadione, demonstrate NADH-driven transport activity. This activity increases linearly with the NADH oxidase activity, but maximal transprt activities are reached under conditions where the NADH oxidase activity is not yet maximal. These results indicate that the rate of energy supply is the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor under conditions of high NADH oxidase activities. Under energy-limiting conditions 135-235 molecules of NADH have to be oxidized in order to transport one molecule of amino acid. At all levels of energy supply a competition by the different amino acid transport systems for the available energy could not be observed. These observations indicate that only a fraction of the energy, generated by the respiratory chain, is used for the transport of an amino acid and that the bulk of the energy dissipates via other channels in the membrane vesicles.