Evidence that Na+-pumping occurs through the D-channel in Vitreoscilla cytochrome bo (original) (raw)
Related papers
Role of Asp544 in subunit I for Na+ pumping by Vitreoscilla cytochrome bo
Biochemical and Biophysical Research Communications, 2006
The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H + pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na + pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na + as well as decreased stimulation in respiratory activity in the presence of Na + . Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes.
Role of Asp544 in subunit I for Na{sup +} pumping by Vitreoscilla cytochrome bo
Biochem Biophys Res Commun, 2006
The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H + pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na + pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100, which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na + as well as decreased stimulation in respiratory activity in the presence of Na +. Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes.
Biochemistry, 2009
Vibrio cholerae and many other marine and pathogenic bacteria posses a unique respiratory complex, the Na +-pumping NADH: quinone oxidoreductase (Na +-NQR)1, which pumps Na + across the cell membrane using the energy released by the redox reaction between NADH and ubiquinone. In order to function as a selective sodium pump, Na +-NQR must contain structures that: 1) allow the sodium ion to pass through the hydrophobic core of the membrane, and 2) provide cation specificity to the translocation system. In other sodium transporting proteins, the structures that carry out these roles frequently include aspartate and glutamate residues. The negative charge of these residues facilitates binding and translocation of sodium. In this study we have analyzed mutants of acid residues located in the transmembrane helices of subunits B, D and E of Na +-NQR. The results are consistent with the participation of seven of these residues in the translocation process of sodium. Mutations at NqrB-D397, NqrD-D133 and NqrE-E95 produced a decrease of approximately ten times or more in the apparent affinity of the enzyme for sodium (Km app), which suggests that these residues may form part of a sodium-binding site. Mutation at other residues, including NqrB-E28, NqrB-E144, NqrB-E346 and NqrD-D88, had a large effect on the quinone reductase activity of the enzyme and its sodium sensitivity, but less effect on the apparent sodium affinity, consistent with a possible role in sodium conductance pathways. The sodium pumping NADH:quinone oxidoreductase (Na +-NQR) is a unique prokaryotic respiratory enzyme capable of sustaining a sodium gradient across the plasma membrane, using the free energy released in the coupled oxidation of NADH and reduction of ubiquinone (1-3). Na +-NQR is composed of six subunits (NqrA-F) and contains five cofactors involved in the internal electron transfer: a non-covalently bound FAD and a 2Fe-2S center located in NqrF (4-8), two covalently-bound FMN's in NqrB and NqrC (9-11), which have been shown to give rise to two anionic flavosemiquinone radicals, observed in the partially and fully reduced forms of the enzyme (12), and a non-covalently bound riboflavin molecule that is found as a stable neutral flavosemiquinone radical in the oxidized state of the enzyme (13,14). This is notable because it is the only known instance in which riboflavin is present as a bona fide redox cofactor in any enzyme.
A cytochrome that can pump sodium ion
1990
Previous studies have shown that the bacterium, Vitreoscilla, generates a respiratory-driven AONa+. Two major respiratory electron transport proteins, NADH dehydrogenase (NADH:Quinone oxidoreductase), and cytochrome o terminal oxidase are candidates for the ¢lectrogenic Na + pumping that mediates the AII~Na + formation. The NADH oxidase activity of the membranes was enhanced more by Na + than by Li +. The NADH:Quinone oxidoreductase activity in the respiratory chain was enhanced by Na + and Li +, whereas the quinol oxidase activity of cytochrome _o was enhanced specifically by Na +, and not by Li +, K +, or choline. Purified cytochrome o, reconstituted into Na +loaded liposomes in the right-side-out orientation, catalyzed a net Na + extrusion when energized with Q1H21. In nonloaded inside-out proteoliposomes, this cytochrome catalyzed a net uptake of 22Na+ when energized with ascorbate/TMPD. Both Na+-pumping activities were inhibited by CN-. These results are consistent with the Vitreoscilla cytochrome o being a redox-driven Na + pump.
Energy transducing redox steps of the Na + -pumping NADH:quinone oxidoreductase from Vibrio cholerae
Proceedings of the National Academy of Sciences, 2010
Na + -NQR is a unique respiratory enzyme that couples the free energy of electron transfer reactions to electrogenic pumping of sodium across the cell membrane. This enzyme is found in many marine and pathogenic bacteria where it plays an analogous role to the H + -pumping complex I. It has generally been assumed that the sodium pump of Na + -NQR operates on the basis of thermodynamic coupling between reduction of a single redox cofactor and the binding of sodium at a nearby site. In this study, we have defined the coupling to sodium translocation of individual steps in the redox reaction of Na + -NQR. Sodium uptake takes place in the reaction step in which an electron moves from the 2Fe-2S center to FMN C , while the translocation of sodium across the membrane dielectric (and probably its release into the external medium) occurs when an electron moves from FMN B to riboflavin. This argues against a single-site coupling model because the redox steps that drive these two parts of the...
K+-dependent Na+ transport driven by respiration in Escherichia coli cells and membrane vesicles
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996
Respiration-driven Na + transport from Escherichia coli cells and right-side-out membrane vesicles is strictly dependent on K +. Cells from an E. coli mutant deficient in three major K + transport systems were incapable of accumulating K + or expelling Na + unless valinomycin was added. Membrane vesicles from an E. coli mutant from which the genes encoding the two known electrogenic Na+/nH + antiporters nhaA and nhaB were deleted transported Na + as well as did vesicles from wild-type cells. Quantitative analysis of A~b and ApH showed a high driving force for electrogenic Na+/nH + antiport whether K + was present or not, although Na + transport occurred only in its presence. These results suggest that an Na+/nH + antiporter is not responsible for the Na + transport. Respiration-driven effiux of Na + from vesicles was found to be accompanied by primary uphill effiux of K +. Also, no respiration-dependent effiux of K + was observed in the absence of Na +. Such coupling between Na + and K + fluxes may be explained by the operation of an Na +, K+/H + antiporter previously described in E. coli membrane vesicles (Verkhovskaya, M.L., Verkhovsky, M.I. and WikstrSm, M. (1995) FEBS Lett. 363, 46-48). Active Na + transport is abolished when A~H~ is eliminated by a protonophore, but at low concentrations the protonophore actually accelerated Na + transport. Such an effect may be expected if the Na +, K+/H + antiporter normally operates in tight conjunction with respiratory chain complexes, thus exhibiting some phenomenological properties of a primary redox-linked sodium pump.
Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010
The Na + -translocating NADH:ubiquinone oxidoreductase (Na + -NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na + -NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na + instead of H + . Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na +translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
is a unique energy-transducing complex, widely distributed among marine and pathogenic bacteria. It converts the energy from the oxidation of NADH and the reduction of quinone into an electrochemical Na +-gradient that can provide energy for the cell. Na +-NQR is not homologous to any other respiratory protein but is closely related to the RNF complex. In this review we propose that sodium pumping in Na +-NQR is coupled to the redox reactions by a novel mechanism, which operates at multiple sites, is indirect and mediated by conformational changes of the protein. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).