Characterization of the interaction of NADH with proton pumping E. coli transhydrogenase reconstituted in the absence and in the presence of bacteriorhodopsin

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The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4 -maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the ␣-subunit and nine in the ␤-subunit), with the C terminus of the ␣-subunit and the N terminus of the ␤-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.Nicotinamide-nucleotide transhydrogenases are integral membrane proteins that catalyze the reduction of NADP ϩ by NADH with a concomitant translocation of protons across the membrane, e.g. from the periplasm or intermembrane space to the cytosol or matrix in bacteria and mitochondria, respectively, according to the following:Transhydrogenases are conformationally driven proton pumps in which binding and release of NADP(H) are thought to cause the structural changes allowing hydride transfer as well as proton translocation to occur (1). It is generally concluded that the H ϩ /H Ϫ ratio of the transhydrogenase reaction is 1 (2-4). At present, transhydrogenases from 12 different species have been cloned and/or sequenced that show an overall amino acid sequence identity of 23%. All transhydrogenases possess the same structural organization, regardless of primary sequence arrangement, with three major domains: two hydrophilic domains (domains I and III) comprising a 400-residue-long NAD(H)-binding domain and a 200-residue-long NADP(H)-binding domain, respectively, and domain II, containing a 360-residue-long hydrophobic domain that constitutes the membrane-spanning part of the enzyme (for reviews, see Refs. 4 and 5). The hydrophilic domains have been shown to protrude into the cytosol or matrix in bacteria and mitochondria, respectively (6, 7).Escherichia coli transhydrogenase consists of two subunits, ␣ (510 residues) and ␤ (464 residues), assembled as an ␣ 2 ␤ 2 -tetramer. The membrane domain is composed of the 110 Cterminal residues of the ␣-subunit and the 260 N-terminal residues of the ␤-subunit and ...

Section: Resultsmentioning

confidence: 99%

The Membrane Topology of Proton-pumping Escherichia coli Transhydrogenase Determined by Cysteine Labeling

Meuller

Rydström

1999

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“…It rules out a number of models in which proton translocation was assumed to arise from chemical interactions between nucleotides and a putative hydride-accepting intermediate (reviewed (1)), and it complements our recent evidence that the hydride transfer step itself is not coupled to proton translocation (27). The indications that the coupling reactions involve changes in the nucleotide-binding energy, as suggested by a number of workers (3,28,29), are now very strong. We developed the view that proton translocation is coupled to events that are consequent on the binding and release of NADP ϩ and NADPH (28), and this was supported by the finding that NADP ϩ and NADPH release from domain III are promoted by interactions with the membrane-spanning domain II (16).…”

mentioning

confidence: 93%

Evidence That the Transfer of Hydride Ion Equivalents between Nucleotides by Proton-translocating Transhydrogenase Is Direct

Venning

Grimley

Bizouarn

et al. 1997

Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation.Transhydrogenase is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria. It couples the transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across the membrane. The net reaction is as follows.For many years, the question as to whether hydride transfer between the nucleotides is direct or indirect has been a matter of controversy. It is central to our understanding of the energycoupling reactions. Transhydrogenase comprises three domains. Domains I and III protrude from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria). Domain II spans the membrane. There are separate sites on the enzyme for NAD(H) and for NADP(H); the former is located on domain I, and the latter on domain III (for reviews, see Refs. 1-3).The results of some early experiments on transhydrogenases from mitochondria and from Rhodospirillum rubrum were interpreted as evidence for the existence of a stable, reducedenzyme intermediate (4 -6). It was implied that a functional group on the enzyme, presumably either an amino acid residue or an unidentified prosthetic group, can serve alternately as a hydride acceptor and hydride donor. For example,where E(H) represents the reduced-enzyme intermediate. However, in subsequent work other plausible explanations were found for the earlier data (7, 8). Moreover, the conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7, 9 -11) have been interpreted as evidence that the reaction proceeds through the formation of a ternary complex of enzyme and nucleotide substrates. The addition of nucleotides is random, and fast, relative to the rate of a subsequent step in turnover. The reaction does not appear to take place via a substituted enzyme mechanism, and therefore the existence of a reduced enzyme intermediate, which is stable in the absence of nucleotide, is unlikely (viz. reactions exemplified by Equations 2 and 3). However, the steady-state data do not rule out the possible existence of a reduced enzyme intermediate within the ternary complex, that is a reaction of the following type.The possibility that Cys residues in the polypeptide chain might serve as reducible intermediates in hydride transfer (see, for example, Refs. 1 and 12) has been eliminated by amino acid sequence comparisons, there are no conserved Cys residues in transhydrogenases from different species, and by the fact that complete Cys replacement has only a minimal effect on transhydrogenation activity (13). It is unlikely that other amino acid residues have redox potentials in the appropriate range to serve as intermediates in transhydrogenation between NAD(H) and NADP(H). It ha...

“…Expression and solubilization of the E. coli transhydrogenase enzymes and domains were carried out essentially as described (21). However, cells were resuspended in 30 mM sodium phosphate buffer containing protease inhibitor (Complete TM EDTA-free, Boehringer Mannheim), pH 7.5; cells were disrupted using an X-press (AB Biox, Göteborg, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Mapping of Residues in the NADP(H)-binding Site of Proton-translocating Nicotinamide Nucleotide Transhydrogenase fromEscherichia coli

Fjellström

Axelsson

Bizouarn

et al. 1999

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Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical ␤␣␤␣␤ dinucleotide binding fold is responsible for binding NADP(H). A model defining ␤A, ␣B, ␤B, ␤D, and ␤E of this domain is presented. To test this model, four single cysteine mutants (cf␤A348C, cf␤A390C, cf␤K424C, and cf␤R425C) were introduced into a functional cysteine-free transhydrogenase. Also, five cysteine mutants were constructed in the isolated domain III of Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic characterizations, effects of sulfhydryl-specific labeling with N-ethylmaleimide, 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized 3-aminopyridine adenine dinucleotide (phosphate) were examined.The Membrane-bound transhydrogenase is composed of three domains. In the Escherichia coli enzyme, domain I (ecI, 1 ␣1 to ϳ␣404) and domain III (ecIII, ϳ␤260 to ␤462) are exposed to the cytosol and contain the binding sites for NAD(H) and NADP(H), respectively. Domain II (ϳ␣405 to ␣510 and ␤1 to ϳ␤260) spans the membrane. Domain I (dI) from E. coli (4, 5), Rhodospirillum rubrum (rrI) (6), and bovine (7), and domain III (dIII) from E. coli (5), R. rubrum (8, 9), and bovine (7, 9) have been overexpressed, purified, and partially characterized. So far, domain II has not been expressed as a separate entity. Interestingly, dII is not required for transhydrogenation to occur (decoupled from proton translocation), as initially shown by Yamaguchi and Hatefi (7). Mixtures of recombinant dI and dIII from the same species or from different species catalyze decoupled "forward" and "reverse" reactions (cf. Reaction 1) and the so-called "cyclic reaction" (which involves the reduction of bound NADP ϩ by NADH, followed by the oxidation of bound NADPH by AcPyAD ϩ ) (5,8,9). From a recent study, it was observed that mixtures of rrI plus rrIII and rrI plus ecIII behaved similarly (10). They catalyzed high cyclic reaction rates (about the same as those observed in the complete E. coli and R. rubrum enzymes) that were limited by the transfer of hydride equivalents (10) and slow reverse reaction rates that, with excess rrI under usual assay conditions, were limited by the release of NADP ϩ (5, 8). With this knowledge at hand, it is now possible to use the rrI plus ecIII system to complement mutagenesis experiments performed on the complete enzyme. In addition to substrate binding affinities and hydride equivalent transfer rates, release rates of NADP ϩ and relative affinities between domains are properties that can be studied in mixtures of rrI and ecIII.A three-dimensional model of the NAD(H)-binding site in E.