A change in ionization of the NADP(H)‐binding component (dIII) of proton‐translocating transhydrogenase regulates both hydride transfer and nucleotide release

Rodrigues, Daniel J.; Venning, Jamie D.; Quirk, Philip G.; Jackson, John L.

doi:10.1046/j.1432-1327.2001.02008.x

Cited by 14 publications

(14 citation statements)

References 40 publications

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“…In addition to movement of the entire domain, loop D within domain III also undergoes conformational change. 23,50,51 This is observed in crystals of R. rubrum domain III with two molecules in the asymmetric unit. 27 Loop D displays an open conformation, as seen in the dI 2 dIII complexes, and a closed conformation, in which the entire loop (b subunit residues 396-413) closes over NADPH.…”

Section: Domain III Within Thmentioning

confidence: 75%

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Sundaresan

Chartron

Yamaguchi

et al. 2005

Journal of Molecular Biology

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Section: Domain III Within Thmentioning

confidence: 75%

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Sundaresan

Chartron

Yamaguchi

et al. 2005

Journal of Molecular Biology

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“…The rate constants for release of NADP ϩ and of NADPH from isolated dIII greatly increase at low pH (33). The data suggested that nucleotide release from dIII is preceded by protonation, and the possibility was raised that this might correspond to a step in proton translocation in the intact enzyme.…”

Section: Resultsmentioning

confidence: 98%

“…The change in NADPH fluorescence as it rebound to dIII was negligible. NADP ϩ release from the E155W mutant of dIII following addition of excess NADPH was measured from the change in Trp fluorescence (15,33). The dIII protein (with or without dI) was pre-incubated in 20 mM Mops, pH 7.2, containing 20 M NADP ϩ , at 20°C for 5 min.…”

Section: Methodsmentioning

confidence: 99%

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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“…In the presence of excess NADPH, bound NADP ϩ is displaced from the dIII component on a time scale of a few minutes (27). Table I shows that the effect of exchanging NADP ϩ for NADPH on the phosphorescence lifetime of Trp-72 in dI 2 ⅐dIII 1 complexes is very small.…”

Section: Effects Of Nadh Binding On the Phosphorescence Of Di-di Bindmentioning

confidence: 99%

Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core

Broos

Gabellieri

Boxel

et al. 2003

Journal of Biological Chemistry

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The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component ofRhodospirillum rubrum transhydrogenase are described. This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer. Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions. Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s). The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50°C. In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime. In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site. It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer. The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.Transhydrogenase couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a biological membrane according to Equation 1.The enzyme is found in the inner membrane of higher animal mitochondria and in the cytoplasmic membrane of many bacteria (1). Under physiological conditions, it is thought to utilize the proton electrochemical gradient to generate NADPH. Transhydrogenase comprises three components: dI 1 (ϳ380 residues), which binds NAD ϩ /NADH; dII (ϳ400 residues), the membrane-embedded component harboring the proton translocation channel; and dIII (ϳ200 residues), which binds NADP ϩ / NADPH (Fig. 1). Transhydrogenase is a "dimer" of two dI-dIIdIII "monomers", though the polypeptide composition varies in different species. One of the best characterized transhydrogenases is that from Rhodospirillum rubrum. Recombinant dI and dIII from this organism are stable proteins that bind their cognate nucleotides. Upon mixing, they readily form a dI 2 ⅐dIII 1 complex (K d Ϸ 25 nM), which catalyzes rapid hydride transfer between bound nucleotides. Crystal structures are available for isolated dI (2, 3), dIII (4, 5), and for the complex (6).In transhydrogenase, proton translocation is linked to the redox reaction by way of inter-domain conformational changes. Our current working model is that proton translocation through dII drives transhydrogenase between an "open" state, in which the nucleo...

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A change in ionization of the NADP(H)‐binding component (dIII) of proton‐translocating transhydrogenase regulates both hydride transfer and nucleotide release

Cited by 14 publications

References 40 publications

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

Conformational Diversity in NAD(H) and Interacting Transhydrogenase Nicotinamide Nucleotide Binding Domains

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

Tryptophan Phosphorescence Spectroscopy Reveals That a Domain in the NAD(H)-binding Component (dI) of Transhydrogenase from Rhodospirillum rubrum Has an Extremely Rigid and Conformationally Homogeneous Protein Core

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