Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Phelps, Donna C.; Hatefi, Youssef

doi:10.1021/bi00335a017

Cited by 33 publications

(14 citation statements)

References 24 publications

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Section: Transhydrogenase Couples the Redox Reaction Between Nadh Andsupporting

confidence: 69%

“…This was taken to suggest that transhydrogenase operates by an "alternating-sites" mechanism in which the two dIdII-dIII monomers run 180 o out-of-phase during catalytic turnover. Consistent with this, earlier work had indicated halfof-the-sites inhibition of bovine transhydrogenase by covalent modifiers (21,22).The alternating sites mechanism requires that events in the two dI-dII-dIII monomers are coordinated; there must be a coupling of conformational changes across the dimer interface. In this report, we describe the effects of substituting invariant Tyr 146 , a residue which is centrally located in the dimer interface between the two dI components of R. rubrum transhydrogenase (see Fig.…”

mentioning

confidence: 62%

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Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Obiozo

Brondijk

White

et al. 2007

Journal of Biological Chemistry

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Transhydrogenase couples the redox reaction between NADH and NADP؉ to proton translocation across a membrane. The protein has three components: dI binds NADH, dIII binds NADP ؉ , and dII spans the membrane. Transhydrogenase is a "dimer" of two dI-dII-dIII "monomers"; x-ray structures suggested that the two catalytic sites alternate during turnover. Invariant Tyr 146 in recombinant dI of Rhodospirillum rubrum transhydrogenase was substituted with Phe and Ala (proteins designated dI.Y146F and dI.Y146A, respectively). Analytical ultracentrifuge experiments and differential scanning calorimetry show that dI.Y146A more readily dissociates into monomers than wild-type dI. Analytical ultracentrifuge and Trp fluorescence experiments indicate that the dI.Y146A monomers bind NADH much more weakly than dimers. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics. However, dI.Y146A reconstituted activity in its dimeric form but not in its monomeric form, this despite monomers retaining their native fold and binding to the dI-depleted membranes. It is suggested that transhydrogenase reconstructed with monomers of dI.Y146A is catalytically compromised, at least partly as a consequence of the lowered affinity for NADH, and this results from lost interactions between the nucleotide binding site and the protein ␤-hairpin upon dissociation of the dI dimer. The importance of these interactions and their coupling to dI domain rotation in the mechanism of action of transhydrogenase is emphasized. Two peaks in the 1 H NMR spectrum of wild-type dI are broadened in dI.Y146A and are tentatively assigned to S-methyl groups of Met resonances in the ␤-hairpin, consistent with the segmental mobility of this feature in the structure.Transhydrogenase is located in the inner mitochondrial membrane of animal cells and in the cytoplasmic membrane of bacteria. It catalyzes the reduction of NADP ϩ by NADH coupled to the translocation of protons across the membrane shown in Reaction 1.The enzyme provides NADPH for biosynthesis reactions (1, 2) and for glutathione reduction in the protection of cells against oxidative stress (3-5). A role for transhydrogenase has also been implicated in glucose-stimulated insulin secretion by pancreatic ␤-cells (6).Transhydrogenase has three components. The dI component, which binds NADH, and the dIII component, which binds NADP ϩ , protrude from the membrane, and the dII component spans the membrane. Conformational changes link the redox reaction at the dI/dIII interface with proton translocation through dII (for recent reviews, see Refs. 7,8). The intact enzyme is a "dimer" of two dI-dII-dIII "monomers" (9 -12), although the disposition of dI, dII, and dIII along polypeptides is somewhat variable between species (13). Solution studies (14, 15) and x-ray structures (16 -20) reveal the profound asymmetry of a complex formed from mixtures of dI and dIII (the socalled dI 2 dIII 1 complex) of the Rhodospirillum rubrum enzyme. This was taken to suggest that transhydrogen...

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Section: Transhydrogenase Couples the Redox Reaction Between Nadh Andsupporting

confidence: 69%

mentioning

confidence: 62%

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Obiozo

Brondijk

White

et al. 2007

Journal of Biological Chemistry

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“…Because hydride transfer across the single dI/dIII interface of the complex is extremely rapid (25), it was proposed that in the complete enzyme the two dI/dIII interfaces must be alternately brought together during turnover. The proposal is supported by earlier studies indicating "half of the sites" reactivity for bovine transhydrogenase (26,27) and by another investigation in which inactivation of the bovine enzyme by high concentrations of Triton X-100 was attributed to the dissociation of dimers into monomers (18).…”

mentioning

confidence: 52%

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

Weston¹,

Venning

Jackson

2002

Journal of Biological Chemistry

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Unlike their bacterial and mammalian counterparts, the NADP(H)-and NAD(H)-binding components of proton-translocating transhydrogenase from the protozoan parasite Entamoeba histolytica (denoted ehdIII and ehdI, respectively) are tethered by a polypeptide linker. The recombinant tethered fragment, ehdIII-ehdI, was prepared without its membrane-spanning dII component. Dimers of ehdIII-ehdI catalyzed transhydrogenation, but monomers were inactive. The addition of ehdIII to ehdIIIehdI monomers did not lead to an increase in the rate of transhydrogenation, showing that this inactivity is not the result of an unfavorable topology introduced by the linker. The addition of a bacterial dI to ehdIII-ehdI led to an increase in the rate of transhydrogenation, showing that the linker is flexible. A hybrid protein in which ehdIII is tethered to the bacterial dI (denoted ehdIII-rrdI) more readily formed active dimers. Data from small angle x-ray scattering by the hybrid dimers were fitted to models derived from the high-resolution crystal structure of the bacterial dI 2 Some membrane proteins, through changes in their conformation, can link a scalar chemical reaction to the translocation of solutes or ions. Often in these proteins, oligomeric interactions are central to the mechanism. For example, the F 1 F oATPase operates by a 3-site rotary mechanism (1) and the P-glycoprotein undergoes a 2-site alternation during turnover (2). Although it catalyzes a redox reaction rather than ATP hydrolysis, transhydrogenase is in this category of proteins, and its amenable properties make it an excellent model for studying conformational coupling (reviewed in Refs. 3 and 4). The enzyme links the hydride transfer between NAD(H) and NADP(H) to the translocation of protons across the inner membranes of mammalian mitochondria and the cytoplasmic membranes of bacteria as shown in Reaction 1.The reaction is driven from left to right by the proton electrochemical gradient generated by the respiratory (or sometimes photosynthetic) electron transport chain. Thus, transhydrogenase is important as a source of NADPH for biosynthesis and glutathione reduction (for protection against free-radical damage) and in the regulation of flux through the tricarboxylic acid cycle (5, 6). Genes encoding transhydrogenase have also been found in several protozoan parasites. In Plasmodium falciparum the enzyme is probably located in the mitochondrial membrane, but in Eimeria tenella it is thought to be associated with so-called "refractile bodies" (7) and in Entamoeba histolytica, with "mitosomes" (8), otherwise known as "cryptons" (9). The functions of the Ei. tenella refractile bodies and the En. histolytica mitosomes and the nature of the partner proteins that are coupled to the transhydrogenase in the local chemiosmotic proton circuits are not known. The partner proteins are probably not enzymes normally associated with oxidative phosphorylation (10). All proton-translocating transhydrogenases seem to have a similar structural organization. There is a dII compon...

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“…First, p-fluorosulfonylbenzoyl 5'-adenosine (FS0,BzAdo) inhibits transhydrogenase from both bovine mitochondria [36] and E. coli [34]. NADH and its analogues protect against the inhibition.…”

Section: Changes In the Conformation Of The Mobile Loop Upon Binding mentioning

confidence: 99%

Conformational Dynamics of a Mobile Loop in the NAD(H)‐Binding Subunit of Proton‐Translocating Transhydrogenases from Rhodospirillum Rubrum and Escherichia Coli

Diggle¹,

Cotton²,

Grimley³

et al. 1995

European Journal of Biochemistry

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Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Th,) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Th, in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Th, specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the onedimensional 'H-NMR spectra of Th, before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Th, (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Th, with NAD' provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Th,. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

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Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Cited by 33 publications

References 24 publications

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

Substitution of Tyrosine 146 in the dI Component of Proton-translocating Transhydrogenase Leads to Reversible Dissociation of the Active Dimer into Inactive Monomers

The Membrane-peripheral Subunits of Transhydrogenase fromEntamoeba histolytica Are Functional Only When Dimerized

Conformational Dynamics of a Mobile Loop in the NAD(H)‐Binding Subunit of Proton‐Translocating Transhydrogenases from Rhodospirillum Rubrum and Escherichia Coli

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