Structure and mechanism of proton‐translocating transhydrogenase

Jackson, John L.; Peake, Sarah J.; White, Scott A.

doi:10.1016/s0014-5793(99)01644-0

Cited by 52 publications

(64 citation statements)

References 77 publications

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“…These conformational changes are coupled to proton translocation across the membrane. They are propagated in the membrane-spanning dII and are associated with changes in NADP ϩ and NADPH binding to dIII (22). Thus, dIII may adopt an "open" state, in which bound NADP(H) can readily exchange with nucleotides in the solvent, and an "occluded" state, in which this exchange is blocked.…”

Section: Discussionmentioning

confidence: 99%

“…Those from R. rubrum have been studied in the most detail (11)(12)(13)(14), but the equivalent components from E. coli (12,15), bovine (16,17), human (18), and Entamoeba histolytica (19) transhydrogenases have similar properties. High resolution structures of both dI (20) and dIII (21)(22)(23)(24) have now been published. Remarkably, a simple mixture of dI and dIII proteins, even from different species, forms a complex that catalyzes transhydrogenation.…”

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confidence: 99%

“…On the basis of the crystal structures and the observations on the behavior of the complex of dI and dIII in solution, we proposed an alternating-site model for intact transhydrogenase; conformational changes resulting from proton translocation across the membrane give rise to changes in interaction between dIII and dI alternately in the two halves of the molecule, and these drive the nucleotide-binding changes that are responsible for coupling to the redox reaction (3,22). In the experiments described below, we provide evidence for the stability of a dI 2 dIII 1 heterotrimer in solution conditions, and we show that the binding of nucleotides to isolated dI and dIII is affected by heterotrimer formation.…”

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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: Discussionmentioning

confidence: 99%

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confidence: 99%

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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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“…The relative simplicity of transhydrogenase, the emergence of methods for determining its rate of reaction in real time (3)(4)(5)(6), and recent high resolution structural information (7)(8)(9)(10)(11)(12) make it a good model for understanding the general principles of operation of conformationally coupled ion translocators. The enzyme is composed of three components.…”

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confidence: 99%

“…We proposed an NADP(H) binding change model in which NADP ϩ (or NADPH) from the solvent can only bind to (or leave from) an "open" state of the dIII component of the protein and in which the redox reaction can only take place in an "occluded" state. Association and dissociation of protons during translocation, gated by the redox state of the NADP(H), drives the protein between the open and occluded states (16,7). Recent observations on pronounced structural asymmetries in transhydrogenase suggest that the two dI⅐dII⅐dIII trimers of the complete enzyme undergo reciprocating alternations of conformation during turnover (12).…”

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Fast Hydride Transfer in Proton-translocating Transhydrogenase Revealed in a Rapid Mixing Continuous Flow Device

Pinheiro¹,

Venning²,

Jackson³

2001

Journal of Biological Chemistry

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Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Coupling is achieved through changes in protein conformation. Upon mixing, the isolated nucleotide-binding components of transhydrogenase (dI, which binds NAD(H), and dIII, which binds NADP(H)) form a catalytic dI 2 ⅐dIII 1 complex, the structure of which was recently solved by x-ray crystallography. The fluorescence from an engineered Trp in dIII changes when bound NADP ؉ is reduced. Using a continuous flow device, we have measured the Trp fluorescence change when dI 2 ⅐dIII 1 complexes catalyze reduction of NADP ؉ by NADH on a sub-millisecond scale. At elevated NADH concentrations, the first-order rate constant of the reaction approaches 21,200 s ؊1 , which is larger than that measured for redox reactions of nicotinamide nucleotides in other, soluble enzymes. Rather high concentrations of NADH are required to saturate the reaction. The deuterium isotope effect is small. Comparison with the rate of the reverse reaction (oxidation of NADPH by NAD ؉ ) reveals that the equilibrium constant for the redox reaction on the complex is >36. This high value might be important in ensuring high turnover rates in the intact enzyme.Transhydrogenase is found in the inner membranes of animal mitochondria and in the cytoplasmic membranes of many bacteria. It couples the redox reaction between NAD(H) and NADP(H) to inward translocation of protons across the membrane (Eq. 1).The enzyme provides NADPH for biosynthesis and glutathione reduction, and in mitochondria, it also helps to control flux through the tricarboxylic acid cycle (1, 2). The relative simplicity of transhydrogenase, the emergence of methods for determining its rate of reaction in real time (3-6), and recent high resolution structural information (7-12) make it a good model for understanding the general principles of operation of conformationally coupled ion translocators. The enzyme is composed of three components. dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude into the mitochondrial matrix (or the bacterial cytoplasm), and dII spans the membrane. The intact enzyme is effectively a dimer of two dI⅐dII⅐dIII "trimers" (13,14), though there are species variations in the way the polypeptide chains are joined. The findings that the transfer of hydride-ion equivalents between the bound nucleotides on transhydrogenase is direct (3, 5) and that there is no exchange of the transferred hydride with water protons (15) together establish that coupling to proton translocation does not occur at the redox step. We proposed an NADP(H) binding change model in which NADP ϩ (or NADPH) from the solvent can only bind to (or leave from) an "open" state of the dIII component of the protein and in which the redox reaction can only take place in an "occluded" state. Association and dissociation of protons during translocation, gated by the redox state of the NADP(H), drives the protein between the open and occluded states (16, 7). Recent observations on pro...

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Archaeal lipid hydrogen isotopes in a marine thaumarchaeon

Leavitt

Kopf

Weber

et al. 2022

Preprint

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The stable hydrogen isotope composition of persistent biomolecules is used as a paleoenvironmental proxy. While much previous work has focused on plant leaf wax-derived n-alkanes, the potential of bacterial and archaeal lipid biomarkers as carriers of H isotope signatures remains underexplored. Here we investigated H isotope distributions in the membrane lipids of the ammonia-oxidizing chemoautotroph Nitrosopumilus maritimus strain SCM1. Hydrogen isotope ratios were measured on the biphytane chains of tetraether membrane lipids extracted from steady-state continuous cultures cultivated at slow, medium, and fast growth rates. In contrast to recent work on bacterial fatty acids, where the direction and magnitude of isotopic fractionation varies widely (ca. 600 energy metabolism, archaeal biphytane data in the present work are relatively invariant. The weighted average 2H/1H fractionation values relative to growth water (2εL/W) only ranged from 272 to 260 a three-fold difference in doubling times (30.8 hr to 92.5 hr), yielding an average growth-rate effect of 0.2 depleted than all heterotrophic archaeal and bacterial lipid H isotope measurements in the literature, and on par with those from other autotrophic archaea, as well as isoproenoid-based lipids in photoautotrophic algae. N. maritimus values of 2εL/W also varied systematically with the number of internal rings (cyclopentyl + cyclohexyl), increasing for each additional ring by 6.4 ± 2.7 an isotope flux-balance model in tandem with a comprehensive analysis of the sources of H in archaeal lipid biosynthesis, we use this observation to estimate the kinetic isotope effects (KIEs) of H incorporation from water; from reducing cofactors such as ferredoxin, and for the transhydrogenation reaction(s) that convert the electron-donor derived NADH into NADPH for anabolic reactions. Consistent with prior studies on bacteria, our results indicate the KIEs of reducing cofactors and transhydrogenation processes in archaea are highly fractionating, while those involving exchange of water protons are less so. When combined with the observation of minimal growth-rate sensitivity, our results suggest biphytanes of autotrophic 3HP/4HB Thaumarchaeota may be offset from source waters by a nearly constant 2εL/W value. Together with the ring effect, this implies that all biphytanes originating from a common source should have a predictable ordering of their isotope ratios with respect to biphytane ring number, allowing precise reconstruction of the original δ2H value of the growth water. Collectively, these patterns indicate archaeal biphytanes have potential as paleo-hydrological proxies, either as a complement or an alternative to leaf wax n-alkanes.

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Structure and mechanism of proton‐translocating transhydrogenase

Cited by 52 publications

References 77 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

Fast Hydride Transfer in Proton-translocating Transhydrogenase Revealed in a Rapid Mixing Continuous Flow Device

Archaeal lipid hydrogen isotopes in a marine thaumarchaeon

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