Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH

NADH:quinone oxidoreductase, or Complex I, is a multi-subunit membrane-bound enzyme in the respiratory chain of many pro- and eukaryotes. The enzyme catalyzes the oxidation of NADH and donates electrons to the quinone pool, coupled to proton translocation across the membrane, but the mechanism of energy transduction is not understood. In bacteria the enzyme consists of 14 subunits, seven membrane spanning and seven protruding from the membrane. The hydrophobic NuoH (NQO8, ND1, NAD1, NdhA) subunit is seemingly involved in quinone binding. A homologous, structurally and most likely functionally similar subunit is also found in F(420)H2 oxidoreductases and in complex membrane-bound hydrogenases. We have made theoretical analyses of NuoH and NuoH-like polypeptides and experimentally analyzed the transmembrane topology of the NuoH subunit from Rhodobacter capsulatus by constructing and analyzing alkaline phosphatase fusion proteins. This demonstrated that the NuoH polypeptide has eight transmembrane segments, and four highly conserved hydrophilic sequence motifs facing the inside, bacterial cytoplasm. The N-terminal and C-terminal ends are located on the outside of the membrane. A topology model of NuoH based on these results is presented, and implications from the model are discussed.     

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.     

Phylogenomic analysis of type 1 NADH:Quinone oxidoreductase

We performed phylogenomic analysis of the catalytic core of NADH:quinone oxidoreductases of type 1 (NDH-1). Analysis of phylogenetic trees, as constructed for the core subunits of NDH-1, revealed fundamental differences in their topologies. In the case of four putatively homologous ion-carrying membrane subunits, the trees for the NuoH and NuoN subunits contained separate archaeal clades, whereas subunits NuoL and NuoM were characterized by multiple archaeal clades spread among bacterial branches. Large, separate clades, which united sequences belonging to different archaeal subdomains, were also found for cytoplasmic subunits NuoD and NuoB, homologous to the large and small subunits of nickel-iron hydrogenases. A smaller such clade was also shown for subunit NuoC. Based on these data, we suggest that the ancestral NDH-1 complex could be present already at the stage of the Last Universal Cellular Ancestor (LUCA). Ancestral forms of membrane subunits NuoN and NuoH and cytoplasmic subunits NuoD, NuoB, and, perhaps NuoC, may have formed a membrane complex that operated as an ion-translocating membrane hydrogenase. After the complex attained the ability to reduce membrane quinones, gene duplications could yield the subunits NuoL and NuoM, which enabled translocation of additional ions.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues

Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.     

N,N'-dicyclohexylcarbodiimide-binding proteolipid of the vacuolar H+-ATPase from oat roots

The inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was used to probe the structure and function of the vacuolar H+-translocating ATPase from oat roots (Avena sativa var. Lang). The second-order rate constant for DCCD inhibition was inversely related to the concentration of membrane, indicating that DCCD reached the inhibitory site by concentrating in the hydrophobic environment. [14C]DCCD preferentially labeled a 16-kDa polypeptide of tonoplast vesicles, and the amount of [14C]DCCD bound to the 16-kDa peptide was directly proportional to inhibition of ATPase activity. A 16-kDa polypeptide had previously been shown to be part of the purified tonoplast ATPase. As predicted from the observed noncooperative inhibition, binding studies showed that 1 mol of DCCD was bound per mol of ATPase when the enzyme was completely inactivated. The DCCD-binding 16-kDa polypeptide was purified 12-fold by chloroform/methanol extraction. This protein was thus classified as a proteolipid, and its identity as part of the ATPase was confirmed by positive reaction with the antibody to the purified ATPase on immunoblots. From the purification studies, we estimated that the 16-kDa subunit was present in multiple (4-8) copies/holoenzyme. The purification of the proteolipid is a first step towards testing its proposed role in H+ translocation.     

Modeling of human pathogenic mutations in Escherichia coli complex I reveals a sensitive region in the fourth inside loop of NuoH

Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations.Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K_m value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.     

The Origin of Cluster N2 of the Energy-Transducing NADH–Quinone Oxidoreductase: Comparisons of Phylogenetically Related Enzymes

NADH–quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient ( ). Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.     

Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pivotal roles of three conserved carboxyl residues of the NuoC (30k) segment in the structural integrity of proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The prokaryotic proton-translocating NADH-quinone oxidoreductase (NDH-1) is an L-shaped membrane-bound enzyme that contains 14 subunits (NuoA-NuoN or Nqo1-Nqo14). All subunits have their counterparts in the eukaryotic enzyme (complex I). NDH-1 consists of two domains: the peripheral arm (NuoB, -C, -D, -E, -F, -G, and -I) and the membrane arm (NuoA, -H, -J, -K, -L, -M, and -N). In Escherichia coli NDH-1, the hydrophilic subunits NuoC/Nqo5/30k and NuoD/Nqo4/49k are fused together in a single polypeptide as the NuoCD subunit. The NuoCD subunit is the only subunit that does not bear a cofactor in the peripheral arm. While some roles for inhibitor and quinone association have been reported for the NuoD segment, structural and functional roles of the NuoC segment remain mostly elusive. In this work, 14 highly conserved residues of the NuoC segment were mutated and 21 mutants were constructed using the chromosomal gene manipulation technique. From the enzymatic assays and immunochemical and blue-native gel analyses, it was found that residues Glu-138, Glu-140, and Asp-143 that are thought to be in the third α-helix are absolutely required for the energy-transducing NDH-1 activities and the assembly of the whole enzyme. Together with available information for the hydrophobic subunits, we propose that Glu-138, Glu-140, and Asp-143 of the NuoC segment may have a pivotal role in the structural stability of NDH-1.     

Quinone binding and reduction by respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) has a central function in oxidative phosphorylation and hence for efficient ATP production in most prokaryotic and eukaryotic cells. This huge membrane protein complex transfers electrons from NADH to ubiquinone and couples this exergonic redox reaction to endergonic proton pumping across bioenergetic membranes. Although quinone reduction seems to be critical for energy conversion, this part of the reaction is least understood. Here we summarize and discuss experimental evidence indicating that complex I contains an extended ubiquinone binding pocket at the interface of the 49-kDa and PSST subunits. Close to iron–sulfur cluster N2, the proposed immediate electron donor for ubiquinone, a highly conserved tyrosine constitutes a critical element of the quinone reduction site. A possible quinone exchange path leads from cluster N2 to the N-terminal β-sheet of the 49-kDa subunit. We discuss the possible functions of a highly conserved HRGXE motif and a redox–Bohr group associated with cluster N2. Resistance patterns observed with a large number of point mutations suggest that all types of hydrophobic complex I inhibitors also act at the interface of the 49-kDa and the PSST subunit. Finally, current controversies regarding the number of ubiquinone binding sites and the position of the site of ubiquinone reduction are discussed.     

Inactivation of Rb+ and Na+ occlusion on (Na+,K+)-ATPase by modification of carboxyl groups

This paper demonstrates and characterizes inactivation by N,N'-dicyclohexylcarbodiimide (DCCD) of Rb+ and Na+ occlusion in pig kidney (Na+,K+)-ATPase. Rb+ and Na+ occlusion dependent on oligomycin are measured with a manual assay. Parallel measurement of phosphorylation (by Pi plus ouabain) and Na+ or Rb+ occlusion lead to stoichiometries of 3 Na+ or 2 Rb+ per pump molecule. Inactivation of cation occlusion by DCCD shows the following features: (a) Rb+ and Na+ occlusion are inactivated with identical rates and (b) DCCD concentration dependence shows first-order kinetics and also proportionality to the ratio of DCCD to protein, (c) Rb+ and Na+ occlusion are equally protected from DCCD, by Rb+ ions with high affinity (or Na+ ions with lower affinity), (d) inactivation is only slightly pH-dependent between 6 and 8.5 but (e) is significantly accelerated by several hydrophobic amines while a water-soluble nucleophile, glycine ethyl ester has no effect, and (f) inactivation is exactly correlated with inactivation of (Na+,K+)-ATPase activity of ATP-dependent Na+/K+ exchange in reconstituted vesicles and with the magnitude of E1Na+----E2(Rb+) conformational transitions measured with fluorescence probes. The simplest hypothesis to explain the results is that DCCD modifies one (or a small number of) critical carboxyl residues in a non-aqueous cation binding domain and so blocks occlusion of 2 Rb+ or 3 Na+ ions. The results suggest further that Na+ and K+(Rb+) bind to the same sites and are transported sequentially on the same trans-membrane segments. A second effect of the DCCD treatment is a 4-8-fold shift of the conformational equilibrium E2(Rb+)----E1Rb+ toward E1Rb+. This is detected by (a) reduction in apparent Rb+ affinity for Rb+ occlusion or Rb+/Rb+ exchange in vesicles and (b) direct demonstration of an increased rate of E2(K+)----E1Na+ and decreased rate of E1Na+----E2(K+). This effect is not protected against by Rb+ ions and probably reflects modification of a second group of residues. Modification of (Na+,K+)-ATPase by carbodiimides is complex. Depending on the nature of the carbodiimide (water- or lipid-soluble), ratio of carbodiimide to protein, and perhaps source of the enzyme, inactivation might result either from modification of critical carboxyls, as suggested by this work, or from internal cross-linking as proposed by Pedemonte, C. H. and Kaplan, J. H. ((1986) J. Biol. Chem. 261, 3632-3639).     

Characterization of the 9.5-kDa ubiquinone-binding protein of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa.

A small polypeptide subunit of the NADH:ubiquinone reductase (complex I) from Neurospora crassa has been identified by photoaffinity labeling to participate in the binding of ubiquinone [Heinrich, H., & Werner, S. (1992) Biochemistry (preceding paper in this issue)]. This polypeptide is further characterized by its primary structure and by an assessment of its localization within complex I. A lambda gt11 cDNA expression library was screened using a specific antibody directed against this individual subunit of complex I. Two groups of clones, coding for polypeptide subunits of the appropriate apparent molecular weight, were isolated. One group was shown to contain the relevant recombinants. The derived amino acid sequence for the 9.5-kDa ubiquinone-binding polypeptide shows a similarity with a putative ubiquinol-binding subunit (also a 9.5-kDa polypeptide) from complex III of bovine heart [Usui, S., Yu, L., & Tu, C.-A. (1990) Biochemistry 29, 4618-4626]. The polypeptide has a hydrophobic stretch of a sufficient length to span the membrane. It resists against extraction with NaBr or Na2CO3, and therefore probably is buried in the so-called hydrophobic membrane portion of complex I. This nuclearly-encoded subunit lacks a typical cleavable presequence and is imported into isolated mitochondria by a membrane potential-dependent process.     

The coupling mechanism of respiratory complex I — A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The coupling mechanism of respiratory complex I - A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots

The subunit organization of the tonoplast H+-pumping ATPase from oat roots (Avena sativa L. var. Lang) was investigated. Tonoplast vesicles were treated with low ionic strength solutions (0.1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer or 0.1 mM Na EDTA), carbonate, or a chaotropic reagent (KI), and then centrifuged to give a soluble fraction and a pellet. Treatments with low ionic strength solutions or KI resulted in 70-80% reduction in the membrane-associated ATPase activity, but did not affect the K+-stimulated pyrophosphatase activity. Polypeptides of 72, 60, and 41 kDa were solubilized from tonoplast vesicles by these wash treatments. These polypeptides reacted with polyclonal antibodies against the holoenzyme of tonoplast ATPase (anti-ATPase) and copurified with the tonoplast ATPase activity during gel filtration chromatography (Sepharose CL-6B). Mono-specific antibody against the 72- or 60-kDa polypeptide reacted with the solubilized 72- or 60-kDa polypeptide, respectively. However, the N,N-[14C]dicyclohexylcarbodiimide-binding 16-kDa polypeptide and a 13-kDa polypeptide that also reacted with anti-ATPase and copurified with the tonoplast ATPase activity during gel filtration remained in the pellets after the wash treatments. We conclude that the 72- and 60-kDa polypeptides appear to be peripheral subunits of the tonoplast ATPase and that the 16-kDa polypeptide is probably embedded in the membrane bilayer. Additional subunits of the ATPase complex may include a 41-kDa (peripheral) and a 13-kDa (integral) polypeptide. Based on these results, a working model of the tonoplast ATPase analogous to the F1F0-ATPase is proposed.     

Modification of the catalytic function of the mitochondrial cytochrome b-c1 complex by dicyclohexylcarbodiimide

N,N'-Dicyclohexylcarbodiimide (DCCD) induces a complex set of effects on the succinate-cytochrome c span of the mitochondrial respiratory chain. At concentrations below 1000 mol per mol of cytochrome c1, DCCD is able to block the proton-translocating activity associated to succinate or ubiquinol oxidation without inhibiting the steady-state redox activity of the b-c1 complex either in intact mitochondrial particles or in the isolated ubiquinol-cytochrome c reductase reconstituted in phospholipid vesicles. In parallel to this, DCCD modifies the redox responses of the endogenous cytochrome b, which becomes more rapidly reduced by succinate, and more slowly oxidized when previously reduced by substrates. At similar concentrations the inhibitor apparently stimulates the redox activity of the succinate-ubiquinone reductase. Moreover, DCCD, at concentrations about one order of magnitude higher than those blocking proton translocation, produces inactivation of the redox function of the b-c1 complex. The binding of [14C]DCCD to the isolated b-c1 complex has shown that under conditions leading to the inhibition of the proton-translocating activity of the enzyme, a subunit of about 9500 Da, namely Band VIII, is the most heavily labelled polypeptide of the complex. The possible correlations between the various effects of DCCD and its modification of the b-c1 complex are discussed.     

Dicyclohexylcarbodiimide interaction with sarcoplasmic reticulum. Inhibition of Ca2+ efflux

Dicyclohexylcarbodiimide (DCCD), a hydrophobic carboxyl reagent, inhibited Ca2+ release from Ca2+-loaded sarcoplasmic reticulum vesicles, induced by elevated pH, tetraphenylboron, ATP + Pi, or membrane modification with acetic anhydride. Under the conditions used, the same concentrations of DCCD were required for inhibition of Ca2+ release, Ca2+-ATPase activity, and Ca2+ uptake. On the other hand, free Ca2+ or alkaline pH prevented the inhibition by DCCD of Ca2+-ATPase and coupled Ca2+ transport but not that of Ca2+ release. Moreover, several hydrophilic carboxyl reagents inhibited Ca2+-ATPase but not Ca2+ release. We suggest that a carboxyl residue(s), located in a hydrophobic region of a protein(s), is involved in the control of Ca2+ release, where DCCD interaction with this group blocks Ca2+ release. This group is distinct from the one involved in the inhibition of Ca2+-ATPase. DCCD also inhibited [3H]ryanodine binding to junctional sarcoplasmic reticulum membranes. The presence of Ca2+ or an alkaline pH only slightly affects the degree of inhibition of ryanodine binding by DCCD. Incubation of the membranes with [14C]DCCD resulted in labeling of 350-, 170-, 140-, 53-, and 30-kDa proteins in addition to the Ca2+-ATPase. The involvement of one or all of the DCCD-labeled proteins in Ca2+ release and ryanodine binding is discussed.     

Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.     

The C-terminally truncated NuoL subunit (ND5 homologue) of the Na+-dependent complex I from Escherichia coli transports Na+

The NADH:quinone oxidoreductase (complex I) from Escherichia coli acts as a primary Na+ pump. Expression of a C-terminally truncated version of the hydrophobic NuoL subunit (ND5 homologue) from E. coli complex I resulted in Na+-dependent growth inhibition of the E. coli host cells. Membrane vesicles containing the truncated NuoL subunit (NuoLN) exhibited 2-4-fold higher Na+ uptake activity than control vesicles without NuoLN. Respiratory proton transport into inverted vesicles containing NuoLN decreased upon addition of Na+, but was not affected by K+, indicating a Na+-dependent increase of proton permeability of membranes in the presence of NuoLN. The His-tagged NuoLN protein was solubilized, enriched by affinity chromatography, and reconstituted into proteoliposomes. Reconstituted His6-NuoLN facilitated the uptake of Na+ into the proteoliposomes along a concentration gradient. This Na+ uptake was prevented by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor against Na+/H+ antiporters.