Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.     

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.     

Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.     

Physiological roles of nicotinamide nucleotide transhydrogenase.

From the foregoing considerations, the energy-linked transhydrogenase reaction emerges as a powerful and flexible element in the network of redox and energy interrelationships that integrate mitochondrial and cytosolic metabolism. Its thermodynamic features make it possible for the reaction to respond readily to challenges, either on the side of NADPH utilization or on the side of energy depletion. Yet, the kinetic features are designed to prevent a wasteful input of energy when other sources of reducing equivalents to NADP are available, or to deplete the redox potential of NADPH in other than emergency conditions. By virtue of these characteristics, the energy-linked transhydrogenase can act as an effective buffer system, guarding against an excessive depletion of NADPH, preventing uncontrolled changes in key metabolites associated with NADP-dependent enzymes and calling on the supply of reducing equivalents from NAD-linked substrates only under conditions of high demand for NADPH. At the same time, it can provide an emergency protection against a depletion of energy, especially in situations of anoxia where a supply of reducing equivalents through NADP-linked substrates can be maintained. The flexibility of this design makes it possible that the functions of the energy-linked transhydrogenase vary from one tissue to another and are readily adjustable to different metabolic conditions.     

Resolution and reconstitution of mitochondrial nicotinamide nucleotide transhydrogenase.

Nicotinamide nucleotide transhydrogenase from bovine heart mitochondria was solubilized with cholate and partially purified by ammoniumsulphate fractionation and density gradient centrifugation. Compared to submitochondrial particles this preparation contained less than 10% of oligomycin-sensitive ATPase and cytochromes. When reconstituted with purified mitochondrial phosphatidylcholine, the enzyme catalyzed a reduction of NAD⁺ by NADPH that was stimulated by uncouplers and which showed a concomitent uncoupler-sensitive uptake of the lipophilic anion tetraphenylboron, indicating the generation of a membrane potential. It is concluded that transhydrogenase can energize the vesicles directly without the intervention of ATPase or cytochromes.     

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc₁ complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.     

Affinity chromatography of mitochondrial nicotinamide nucleotide transhydrogenase

The binding of mitochondrial nicotinamide nucleotide transhydrogenase to NAD+ and NADP+ immobilized to agarose through different parts of the nicotinamide nucleotide molecule was investigated. NADP+ bound at the C8 atom in the adenine moiety proved to be the most efficient ligand whereas that bound at the C3 atom of the ribose moiety was relatively inefficient. NAD+ ligands were generally inactive independently of the site of attachment. Previous results suggest, however, that binding to immobilized NAD+ may be influenced by the detergent in which transhydrogenase is dispersed. Binding to neither ligand was affected by the presence of the second substrate.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Purification and properties of the H(+)-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus.

1. H(+)-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity. 2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53,000 and 48,000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants. 3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H(+)-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli. 4. The dependence of the rate of H- transfer by purified H(+)-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5'-AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex. 5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R.R. & Earle, S.R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H(+)-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.     

Mitochondrial nicotinamide nucleotide transhydrogenase: inhibition by ethoxyformic anhydride, dansyl chloride, and pyridoxal phosphate

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH; EC 1.6.1.1) was inactivated by treatment with pyridoxal phosphate, ethoxyformic anhydride (EFA) or dansyl chloride. NADP and NADPH, but not NAD and NADH, protected TH against inhibition by pyridoxal phosphate, and L-lysine reversed this inhibition. The results suggested modification of an essential lysyl residue by pyridoxal phosphate, possibly at the NADP(H) binding site of TH. EFA and dansyl chloride inhibited TH in a similar manner. The effect of pH on the rate of inhibition of TH by EFA and dansyl chloride was the same, and in both cases addition of NADP and particularly NADPH accelerated the rate of inhibition, while addition of NAD or NADH had no effect. Double inhibition studies, using in one experiment dithiothreitol-reversible inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) to protect the thiol groups of TH, and in another experiment lysine-reversible inhibition by pyridoxal phosphate to protect the putative essential lysyl residues of the enzyme, followed in each case by further treatment of the protected TH with EFA or dansyl chloride, suggested that the inhibitions by EFA and dansyl chloride were independent of the inhibitions by 5,5'-dithiobis (2-nitrobenzoic acid) and pyridoxal phosphate. The inhibitors discussed above are interesting, because pyridoxal phosphate is the only reagent known which appears to modify an essential residue in the NADP(H), but not the NAD(H), binding site of TH, and EFA and dansyl chloride are the only inhibitors known which appear to react with essential residues outside the active site of TH. It is possible that EFA and dansyl chloride inhibitions involve modification of essential prototropic residues in the proton translocation domain of the enzyme.     

Energy-linked nicotinamide nucleotide transhydrogenase: hydrodynamic properties and active form of purified and membrane-bound mitochondrial transhydrogenase from beef heart

The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model.     

Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is modified and inhibited by p-fluorosulfonylbenzoyl-5'-adenosine (FSBA). The modification appears to occur at the NAD(H)-binding site when TH alone or TH in the presence of NADPH is incubated with FSBA. However, when this site is protected by NADH, then FSBA inhibits TH more slowly and modifies a different, though specific, site. This second site could be the NADP(H)-binding site. Using [3H]FSBA in the presence of NADPH, the NAD(H)-binding site was modified, and a single tryptic peptide carrying the label was isolated and sequenced. The amino acid sequence of this peptide was Glu-Ser-Gly-Glu-Gly-Gln-Gly-Gly-Tyr*-Ala-Lys. The modified residue was Tyr. The labeled peptide isolated after incubating TH with [3H]FSBA in the presence of NADH could not be completely purified. However, amino acid analysis and partial sequencing made it possible to identify this segment on the amino acid sequence of bovine TH as derived from its cDNA by Yamaguchi et al. (private communication).     

Regulatory properties of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa. Active enzyme ultracentrifugation studies.

Active enzyme ultracentrifugation studies of the pyridine nucleotide transhydrogenase from Pseudomonas aeruginosa (EC 1.6.1.1.) show that the enzymatic reaction is catalyzed by a molecular species characterized by an S20,W value of about 34 S, whatever the reduced substrate may be (tri- or diphosphopyridine nucleotide). The filamentous aggregated form of the enzyme (S20,W = 121 S and higher), identified by previous investigations (Cohen, P. T., and Kaplan, N. O. (1970), J. Biol. Chem. 245, 2825-2836; Louie, D. D., Kaplan, N. O., and Mc Lean, J. D. (1972), J. Mol. Biol. 70, 651-664), appears, therefore, to be an inactive species. The physiological implications of the enzyme are discussed. Several lines of evidence lead to the conclusion that the transhydrogenase might act as an essential link between carbohydrate catabolism and the respiratory chain.