BBA - Bioenergetics (v.1505, #1)
Coupling mechanism of the oxaloacetate decarboxylase Na+ pump by Peter Dimroth; Petra Jockel; Markus Schmid (1-14).
The oxaloacetate decarboxylase Na+ pump consists of subunits α, β and γ, and contains biotin as the prosthetic group. The peripheral α subunit catalyzes the carboxyltransfer from oxaloacetate to the prosthetic biotin group to yield the carboxybiotin enzyme. Subsequently, this is decarboxylated in a Na+-dependent reaction by the membrane-bound β subunit. The decarboxylation is coupled to Na+ translocation from the cytoplasm into the periplasm, and consumes a periplasmically derived proton. The γ subunit contains a Zn2+ metal ion which may be involved in the carboxyltransfer reaction. It is proposed to insert with its N-terminal α-helix into the membrane and to form a complex with the α subunit with its water-soluble C-terminal domain. The β subunit consists of nine transmembrane α-helices, a segment (IIIa) which inserts from the periplasm into the membrane but does not penetrate it, and connecting hydrophilic loops. The most highly conserved regions of the molecule are segment IIIa and transmembrane helix VIII. Functionally important residues are D203 (segment IIIa), Y229 (helix IV) and N373, G377, S382 and R389 (helix VIII). The polar of these amino acids may constitute a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented translocation of H+ across the membrane. Evidence indicates that two Na+ ions are bound simultaneously to subunit β with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin possibly with the consumption of the proton extracted from S382 and delivered via Y229 to the carboxylated prosthetic group. A conformational change exposes the bound Na+ ions toward the periplasm. With H+ entering from the periplasm, the hydroxyl group of S382 is regenerated, and as a consequence, the Na+ ions are released into this compartment. After switching back to the original conformation, Na+ pumping continues.
Keywords: Oxaloacetate decarboxylase; Na+ pump mechanism, direct coupling; Active site residue;
Sodium ion-translocating decarboxylases by Wolfgang Buckel (15-27).
The review is concerned with three Na+-dependent biotin-containing decarboxylases, which catalyse the substitution of CO2 by H+ with retention of configuration (ΔG°′=−30 kJ/mol): oxaloacetate decarboxylase from enterobacteria, methylmalonyl-CoA decarboxylase from Veillonella parvula and Propiogenium modestum, and glutaconyl-CoA decarboxylase from Acidaminococcus fermentans. The enzymes represent complexes of four functional domains or subunits, a carboxytransferase, a mobile alanine- and proline-rich biotin carrier, a 9–11 membrane-spanning helix-containing Na+-dependent carboxybiotin decarboxylase and a membrane anchor. In the first catalytic step the carboxyl group of the substrate is converted to a kinetically activated carboxylate in N-carboxybiotin. After swing-over to the decarboxylase, an electrochemical Na+ gradient is generated; the free energy of the decarboxylation is used to translocate 1–2 Na+ from the inside to the outside, whereas the proton comes from the outside. At high [Na+], however, the decarboxylases appear to catalyse a mere Na+/Na+ exchange. This finding has implications for the life of P. modestum in sea water, which relies on the synthesis of ATP via ΔμNa+ generated by decarboxylation. In many sequenced genomes from Bacteria and Archaea homologues of the carboxybiotin decarboxylase from A. fermentans with up to 80% sequence identity have been detected.
Keywords: Mechanism of decarboxylation; Sodium ion- and biotin-dependent decarboxylase; Oxaloacetate decarboxylase; Methylmalonyl-CoA decarboxylase; Glutaconyl-CoA decarboxylase; Sodium ion translocation; Bioenergetics;
The Na+-translocating methyltransferase complex from methanogenic archaea by Gerhard Gottschalk; Rudolf K Thauer (28-36).
Methanogenic archaea are dependent on sodium ions for methane formation. A sodium ion-dependent step has been shown to be methyl transfer from N 5-methyltetrahydromethanopterin to coenzyme M. This exergonic reaction (ΔG°′=−30 kJ/mol) is catalyzed by a Na+-translocating membrane-associated multienzyme complex composed of eight different subunits, MtrA–H. Subunit MtrA harbors a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle, demethylation being sodium ion-dependent. Based on the finding that in the cob(II)amide oxidation state the corrinoid is bound in a base-off/His-on configuration it is proposed that methyl transfer from MtrA to coenzyme M is associated with a conformational change of the protein and that this change drives the electrogenic translocation of the sodium ions.
Keywords: Methyltransferase; Sodium ion translocation; Corrinoid protein; Methane formation; Methanogenic archaeon;
Recent progress in the Na+-translocating NADH-quinone reductase from the marine Vibrio alginolyticus by Maki Hayashi; Yuji Nakayama; Tsutomu Unemoto (37-44).
The respiratory chain of Gram-negative marine and halophilic bacteria has a Na+-dependent NADH-quinone reductase that functions as a primary Na+ pump. The Na+-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na+-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.
Keywords: Na+ pump; NADH-quinone reductase; Respiratory chain; Flavin cofactor; Marine bacterium; Vibrio alginolyticus;
Na+ translocation by bacterial NADH:quinone oxidoreductases: an extension to the complex-I family of primary redox pumps 1 1 Dedicated to Peter Dimroth on the occasion of his 60th birthday. by Julia Steuber (45-56).
The current knowledge on the Na+-translocating NADH:ubiquinone oxidoreductase of the Na+-NQR type from Vibrio alginolyticus, and on Na+ transport by the electrogenic NADH:Q oxidoreductases from Escherichia coli and Klebsiella pneumoniae (complex I, or NDH-I) is summarized. A general mode of redox-linked Na+ transport by NADH:Q oxidoreductases is proposed that is based on the electrostatic attraction of a positively charged Na+ towards a negatively charged, enzyme-bound ubisemiquinone anion in a medium of low dielectricity. A structural model of the [2Fe–2S]- and FAD-carrying NqrF subunit of the Na+-NQR from V. alginolyticus based on ferredoxin and ferredoxin:NADP+ oxidoreductase suggests that a direct participation of the Fe/S center in Na+ transport is rather unlikely. A ubisemiquinone-dependent mechanism of Na+ translocation is proposed that results in the transport of two Na+ ions per two electrons transferred. Whereas this stoichiometry of the pump is in accordance with in vivo determinations of Na+ transport by the respiratory chain of V. alginolyticus, higher (Na+ or H+) transport stoichiometries are expected for complex I, suggesting the presence of a second coupling site.
Keywords: Complex I; Na+ transport; NADH; Ubiquinone; Na+/H+ antiporter; Vibrio alginolyticus; Klebsiella pneumoniae;
Structure–function relationships of Na+, K+, ATP, or Mg2+ binding and energy transduction in Na,K-ATPase by Peter L Jorgensen; Per A Pedersen (57-74).
The focus of this article is on progress in establishing structure–function relationships through site-directed mutagenesis and direct binding assay of Tl+, Rb+, K+, Na+, Mg2+ or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E2[2K] or E1P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu327 in transmembrane segment M4, Glu779 in M5, Asp804 and Asp808 in M6 are essential for tight binding of K+ and Na+. Asn324 and Glu327 in M4, Thr774, Asn776, and Glu779 in 771-YTLTSNIPEITP of M5 contribute to Na+/K+ selectivity. Free ATP binding identifies Arg544 as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp710 or Asn713 do not interfere with free ATP binding. Asp710 is essential and Asn713 is important for coordination of Mg2+ in the E1P[3Na] complex, but they do not contribute to Mg2+ binding in the E2P-ouabain complex. Transition to the E2P form involves a shift of Mg2+ coordination away from Asp710 and Asn713 and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp369.
Keywords: Na,K-ATPase; Mutagenesis; Na+ binding; K+ binding; Tl+ binding; Mg2+ binding; ATP binding; Cation binding site; Energy transduction;
Catalytic properties of Na+-translocating V-ATPase in Enterococcus hirae by Takeshi Murata; Miyuki Kawano; Kazuei Igarashi; Ichiro Yamato; Yoshimi Kakinuma (75-81).
V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na+-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na+-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): the ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at the transcriptional level by intracellular Na+ as the signal. Structural aspects and catalytic properties of purified Na+-ATPase closely resembled those of other V-type H+-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na+ at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na+ binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na+ binding sites of the enzyme.
Keywords: Na+-ATPase; V-ATPase; Na+ binding; Na+ binding site; Structure model; Enterococcus hirae;
Na+-driven flagellar motor of Vibrio by Tomohiro Yorimitsu; Michio Homma (82-93).
Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. Bacteria move using rotating helical flagellar filaments. The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane. Flagellar motors are classified into two types according to the coupling ion: namely the H+-driven motor and the Na+-driven motor. Analysis of the flagellar motor at the molecular level is far more advanced in the H+-driven motor than in the Na+-driven motor. Recently, the genes of the Na+-driven motor have been cloned from a marine bacterium of Vibrio sp. and some of the motor proteins have been purified and characterized. In this review, we summarize recent studies of the Na+-driven flagellar motor.
Keywords: Bacterial flagellar motor; Na+-driven motor; Torque generation; Na+ channel blocker; Vibrio alginolyticus;
The Na+-translocating F1F0 ATP synthase of Propionigenium modestum: mechanochemical insights into the F0 motor that drives ATP synthesis 1 1 Dedicated to Peter Dimroth on the occasion of his 60th birthday. by Georg Kaim (94-107).
The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na+ ion gradient. This enzyme clearly belongs to the family of F1F0 ATP synthases and uses exclusively Na+ ions as the physiological coupling ion. The motor domain, F0, comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na+ translocation through F0 proceeds from the periplasm via the stator channel (subunit a) onto a Na+ binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na+ ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F0, which drives the rotation of the γ subunit thus liberating tightly bound ATP from the catalytic sites in F1. We suggest a mechanochemical model for the transduction of transmembrane Na+-motive force into rotary torque by the F0 motor that can account quantitatively for the experimental data.
Keywords: F1F0 ATP synthase; Rotational mechanism; F0 motor; Membrane potential; Na+ occlusion; Propionigenium modestum;
The Na+ cycle in Acetobacterium woodii: identification and characterization of a Na+ translocating F1F0-ATPase with a mixed oligomer of 8 and 16 kDa proteolipids by Volker Müller; Sascha Aufurth; Stefan Rahlfs (108-120).
The homoacetogenic bacterium Acetobacterium woodii relies on a sodium ion current across its cytoplasmic membrane for energy-dependent reactions. The sodium ion potential is established by a yet to be identified primary, electrogenic pump connected to the Wood-Ljungdahl pathway. Reactions possibly involved in Na+ export are discussed. The electrochemical sodium ion potential generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. Biochemical and molecular data identified the Na+-ATPase of A. woodii as a typical member of the F1F0 class of ATPases. Its catalytic properties and the hypothetical sodium ion binding site in subunit c are discussed. The encoding genes were cloned and, surprisingly, the atp operon was shown to contain multiple copies of genes encoding subunit c. Two copies encode identical 8 kDa proteolipids, and a third copy arose by duplication and subsequent fusion of two genes. Furthermore, the duplicated subunit c does not contain the ion binding site in hair pin two. Biochemical and molecular data revealed that all three copies of subunit c constitute a mixed oligomer. The evolution of the structure and function of subunit c in ATPases from eucarya, bacteria, and archaea is discussed.
Keywords: Sodium ion cycle; ATPase; Subunit c; Gene duplication; Mixed oligomer; Acetobacterium woodii;
Sodium-substrate cotransport in bacteria by T.H. Wilson; Ping Z. Ding (121-130).
A variety of sodium-substrate cotransport systems are known in bacteria. Sodium enters the cell down an electrochemical concentration gradient. There is obligatory coupling between the entry of the ion and the entry of substrate with a stoichiometry (in the cases studied) of 1:1. Thus, the downhill movement of sodium ion into the cell leads to the accumulation of substrate within the cell. The melibiose carrier of Escherichia coli is perhaps the most carefully studied of the sodium cotransport systems in bacteria. This carrier is of special interest because it can also use protons or lithium ions for cotransport. Other sodium cotransport carriers that have been studied recently are for proline, glutamate, serine-threonine, citrate and branched chain amino acids.
Keywords: Cotransport; Melibiose; Proline; Glutamate; Serine-threonine; Citrate; Branched chain amino acid;
Towards the molecular mechanism of Na+/solute symport in prokaryotes by Heinrich Jung (131-143).
The Na+/solute symporter family (SSF, TC No. 2.A.21) contains more than 40 members of pro- and eukaryotic origin. Besides their sequence similarity, the transporters share the capability to utilize the free energy stored in electrochemical Na+ gradients for the accumulation of solutes. As part of catabolic pathways most of the transporters are most probably involved in the acquisition of nutrients. Some transporters play a role in osmoadaptation. With a high resolution structure still missing, a combination of genetic, protein chemical and spectroscopic methods has been used to gain new insights into the structure and molecular mechanism of action of the transport proteins. The studies suggest a common 13-helix motif for all members of the SSF according to which the N-terminus is located in the periplasm and the C-terminus is directed into the cytoplasm (except for proteins containing a N- or C-terminal extension). Furthermore, an amino acid substitution analysis of the Na+/proline transporter (PutP) of Escherichia coli, a member of the SSF, has identified regions of particular functional importance. For example, amino acids of TM II of PutP proved to be critical for high affinity binding of Na+ and proline. In addition, it was shown that ligand binding induces widespread conformational alterations in the transport protein. Taken together, the studies substantiate the common idea that Na+/solute symport is the result of a series of ligand-induced structural changes.
Keywords: Secondary transport; Sodium/solute symport; Sodium/proline transporter PutP;
Na+/H+ antiporters by Etana Padan; Miro Venturi; Yoram Gerchman; Nir Dover (144-157).
Na+/H+ antiporters are membrane proteins that play a major role in pH and Na+ homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na+/H+ antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na+/H+ antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 Å resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na+/H+ antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII–IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na+/H+ antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1–G11). The transcription of NhaA has been shown to be regulated by a novel Na+-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.
Keywords: Sodium ion/proton antiporter; Active transport; Membrane protein; Homeostasis of Na+ and H+;
The Na+-dependence of alkaliphily in Bacillus by Terry A. Krulwich; Masahiro Ito; Arthur A. Guffanti (158-168).
A Na+ cycle plays a central role in the remarkable capacity of aerobic, extremely alkaliphilic Bacillus species for pH homeostasis. The capacity for pH homeostasis, in turn, appears to set the upper pH limit for growth. One limb of the alkaliphile Na+ cycle consists of Na+/H+ antiporters that achieve net H+ accumulation that is coupled to Na+ efflux. The major antiporter on which pH homeostasis depends is thought to be the Mrp(Sha)-encoded antiporter, first identified from a partial clone in Bacillus halodurans C-125. Mrp(Sha) may function as a complex. While this antiporter is capable of secondary antiport energized by an imposed or respiration-generated protonmotive force, the possibility of a primary mode has not been excluded. In Bacillus pseudofirmus OF4, at least two additional antiporters, including NhaC, have supporting roles in pH homeostasis. Some of these additional antiporters may be especially important for antiport at low [Na+] or at near-neutral pH. The second limb of the Na+ cycle facilitates Na+ re-entry via Na+/solute symporters and, perhaps, the ion channel associated with the Na+-dependent flagellar motor. The process of pH homeostasis is also enhanced, perhaps especially during transitions to high pH, by different arrays of secondary cell wall polymers in the two alkaliphilic Bacillus species studied most intensively. The mechanisms whereby alkaliphiles handle the challenge of Na+ stress at very elevated [Na+] are just beginning to be identified, and a hypothesis has been advanced to explain the finding that B. pseudofirmus OF4 requires a higher [Na+] for growth at near-neutral pH than at very alkaline pH values.
Keywords: Na+/H+ antiporter; Na+/solute symporter; Na+-motive flagellum; Secondary cell wall polymer;
Role of sodium bioenergetics in Vibrio cholerae by Claudia C. Häse; Blanca Barquera (169-178).
The ability of the bacterium to use sodium in bioenergetic processes appears to play a key role in both the environmental and pathogenic phases of Vibrio cholerae. Aquatic environments, including fresh, brackish, and coastal waters, are an important factor in the transmission of cholera and an autochthonous source. The organism is considered to be halophilic and has a strict requirement for Na+ for growth. Furthermore, expression of motility and virulence factors of V. cholerae is intimately linked to sodium bioenergetics and to each other. Several lines of evidence indicated that the activity of the flagellum of V. cholerae might have an impact on virulence gene regulation. As the V. cholerae flagellum is sodium-driven and the Na+-NQR enzyme is known to create a sodium motive force across the bacterial membrane, it was recently suggested that the increased toxT expression observed in a nqr-negative strain is mediated by affecting flagella activity. It was suggested that the V. cholerae flagellum might respond to changes in membrane potential and the resulting changes in flagellar rotation might serve as a signal for virulence gene expression. However, we recently demonstrated that although the flagellum of V. cholerae is not required for the effects of ionophores on virulence gene expression, changes in the sodium chemical potential are sensed and thus alternative mechanisms, perhaps involving the TcpP/H proteins, for the detection of these conditions must exist. Analyzing the underlying mechanisms by which bacteria respond to changes in the environment, such as their ability to monitor the level of membrane potential, will probably reveal complex interplays between basic physiological processes and virulence factor expression in a variety of pathogenic species.
Keywords: Cholera; Na+-NQR; Sodium motive force; Virulence; Motility; Vibrio cholerae;