Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

Quirk, Philip G.; Jeeves, Mark; Cotton, Nick P.J.; Smith, John K.; Jackson, Baz J.

doi:10.1016/s0014-5793(99)00198-2

Cited by 44 publications

(65 citation statements)

References 25 publications

(44 reference statements)

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“…3), and indirect observations by stopped-flow spectrophotometry (14,34), suggest that binding is quite tight (K d Ͻ 1 M) and this is consistent with a K d Ϸ 25 nM calculated from measurements of catalytic activity (13). NMR experiments reveal an interaction between dI 2 dIII 1 and a second molecule of dIII (25). Significantly, a comparison with the crystal structure shows that some of the amino acid residues involved in this interaction are equivalent to those involved in the interaction of the first dIII with dI 2 within the heterotrimer.…”

Section: Discussionsupporting

confidence: 78%

“…The calculated mass is 20.0 kDa (compare 21.5 kDa from the amino acid sequence of the monomer). NMR experiments with R. rubrum dIII also indicate that the protein is a monomer (25), although on size-exclusion chromatography it behaves anomalously; the apparent M r value is intermediate between those expected for the monomer and the dimer (13). E. coli dIII is monomeric on size-exclusion chromatography (16), but has an unexpectedly large correlation time in NMR experiments, perhaps indicating some tendency to aggregate (37).…”

Section: Resultsmentioning

confidence: 93%

“…A fast kinetic component in the burst, thought to correspond to the transfer of hydride equivalents between nucleotides bound to the complex, requires two dI/dIII for saturation. Second, in NMR experiments, intensity changes in 1 H-15 N cross-peaks from backbone amide groups in R. rubrum dIII upon addition of R. rubrum dI indicate both a slow intermediate and an intermediate exchange between the two proteins (25), and this is taken as evidence for a high affinity (dI 2 ϩ dIII 7 dI 2 dIII 1 ) and a low affinity (dI 2 dIII 1 ϩ dIII 7 dI 2 dIII 2 ) interaction.…”

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confidence: 99%

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The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Venning¹,

Rodrigues²,

Weston³

et al. 2001

Journal of Biological Chemistry

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Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI 2 dIII 1 heterotrimer in solution, but we could find no evidence for the formation of a dI 2 dIII 2 tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotidebinding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI 2 dIII 1 heterotrimer were investigated. (a) The rate of release of NADP ؉ from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI 2 dIII 1 complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.Transhydrogenase, found in the cytoplasmic membranes of bacteria, and in the inner membranes of animal mitochondria, couples the redox reaction between NAD(H) and NADP(H) to the translocation of protons.Its function in energy metabolism, biosynthesis, and detoxification has been discussed at length (1, 2). In different organisms (and possibly in different tissues of the same organism) transhydrogenase can either utilize the proton electrochemical gradient (⌬p) to drive reduction of NADP ϩ by NADH, or it can use NADPH oxidation by NAD ϩ to augment ⌬p formation. Energy coupling in transhydrogenase is indirect. In the forward direction (Reaction 1), protein conformational changes accompanying proton translocation bring together the nicotinamide rings of the bound nucleotides to allow the redox reaction (3). This "binding-change mechanism" may share common features with energy coupling in some ion-translocating ATPases. More generally, transhydrogenase has a number of properties that make it an excellent model for understanding the principles of operation of conformationally linked pumps in biology.The polypeptide organization of transhydrogenases varies between species, but the arrangement of the three components, dI, dII, and dIII, is essentially the same in all (Fig. 1). NAD(H) binds to dI, and NADP(H) binds to dIII; these two components protrude from the membrane (into the bacterial cytoplasm or mitochondrial matrix). The dII component spans the membrane, probably in 13 or 14 transmembrane helices (reviewed in Ref. 4). There is cross-linking and hydrodynamic evidence that both the bovine (5, 6) and Escherichia coli (7) transhydrogenases have two copies each of dI, dII, and dIII...

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Section: Discussionsupporting

confidence: 78%

Section: Resultsmentioning

confidence: 93%

mentioning

confidence: 99%

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The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Venning¹,

Rodrigues²,

Weston³

et al. 2001

Journal of Biological Chemistry

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“…For example, NADPH has a dramatic effect upon the trypsin sensitivity of TH at two remote sites (32,33), and it alters the rate of chemical modification at the NAD(H) site in domain I (2,34). NMR data indicate conformational changes in domain III upon NADP reduction (35). Motion of domain III within TH is described in terms of 'open' and 'occluded' states in the 'alternating site, binding change mechanism' (11).…”

Section: Discussionmentioning

confidence: 99%

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

et al. 2002

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Transhydrogenase (TH) is a dimeric integral membrane enzyme in mitochondria and prokaryotes that couples proton translocation across a membrane with hydride transfer between NAD(H) and NADP(H) in soluble domains. Crystal structures of the NAD(H) binding R1 subunit (domain I) of Rhodospirillum rubrum TH have been determined at 1.8 Å resolution in the absence of dinucleotide and at 1.9 Å resolution with NADH bound. Each structure contains two domain I dimers in the asymmetric unit (AB and CD); the dimers are intimately associated and related by noncrystallographic 2-fold axes. NADH binds to subunits A and D, consistent with the half-of-the-sites reactivity of the enzyme. The conformation of NADH in subunits A and D is very similar; the nicotinamide is in the anti conformation, the A-face is exposed to solvent, and both N7 and O7 participate in hydrogen bonds. Comparison of subunits A and D to six independent copies of the subunit without bound NADH reveals multiple conformations for residues and loops surrounding the NADH site, indicating flexibility for binding and release of the substrate (product). The NADH-bound structure is also compared to the structures of R. rubrum domain I with NAD bound (PDB code 1F8G) and with NAD bound in complex with domain III of TH (PDB code 1HZZ). The NADH-vs NAD-bound domain I structures reveal conformational differences in conserved residues in the NAD(H) binding site and in dinucleotide conformation that are correlated with the net charge, i.e., oxidation state, of the nicotinamides. The comparisons illustrate how nicotinamide oxidation state can affect the domain I conformation, which is relevant to the hydride transfer step of the overall reaction.The energy-transducing nicotinamide nucleotide transhydrogenases of eukaryotic mitochondria and bacteria are homodimeric integral membrane proteins of monomer molecular mass of about 110 kDa. They catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD(H) 1 and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H + /H -stoichiometry of n ) 1 (eq 1) (1-4).In bovine submitochondrial particles, the proton motive force (pmf) accelerates the forward reaction 10-12-fold, and shifts the equilibrium toward product formation. In the reverse direction, transhydrogenation from NADPH to NAD results in outward proton translocation and creation of a pmf. Because there is essentially no difference in the reduction potential of the nicotinamide cofactors, the driving force for proton translocation coupled to the reverse reaction is the difference in binding affinities for substrates (NADPH, NAD) and products (NADH, NADP). In mammalian mitochondria, a function of TH is to produce NADPH for reduction of toxic H 2 O 2 by glutathione reductase and glutathione peroxidase.TH monomers are composed of three domains: a 400-430-residue hydrophilic domain I, a 360-400-residue hydrophobic domain II, and a 200-residue hydrophilic domain III. In mammalian TH, the ...

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“…In crystal structures the equivalent residues to EcbD392 form hydrogen bonds with the pyrophosphate and a ribose hydroxyl group of bound NADP + /NADPH [16,17]. Its unusually high pK a , sensitive to the redox state of the bound nucleotide [35][36][37], and the fact that its substitution results in a failure in NADP + /NADPH binding [33] to isolated dIII, indicate an important catalytic role for this residue.…”

Section: The DI Dii and Diii Components Of Transhydrogenasementioning

confidence: 99%

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

et al. 2015

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Edited by Peter BrzezinskiKeywords: Transhydrogenase Membrane-protein structure Nicotinamide nucleotide Proton-pump Proton-gating a b s t r a c tThe membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP + by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP + /NADPH during enzyme turnover.

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Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

Cited by 44 publications

References 25 publications

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

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Structural changes in the recombinant, NADP(H)‐binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

Cited by 44 publications

References 25 publications

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

The Heterotrimer of the Membrane-peripheral Components of Transhydrogenase and the Alternating-site Mechanism of Proton Translocation

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

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Product

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Crystal Structures of Transhydrogenase Domain I with and without Bound NADH^,