Active-site conformational changes associated with hydride transfer in proton-translocating transhydrogenase

Transhydrogenase couples the redox (hydride-transfer) reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The redox reaction is catalyzed at the interface between two components (dI and dIII) which protrude from the membrane. A complex formed from recombinant dI and dIII (the dI(2)dIII(1) complex) from Rhodospirillum rubrum transhydrogenase catalyzes fast single-turnover hydride transfer between bound nucleotides. In this report we describe three new crystal structures of the dI(2)dIII(1) complex in different nucleotide-bound forms. The structures reveal an asymmetry in nucleotide binding that complements results from solution studies and supports the notion that intact transhydrogenase functions by an alternating site mechanism. In one structure, the redox site is occupied by NADH (on dI) and NADPH (on dIII). The dihydronicotinamide rings take up positions which may approximate to the ground state for hydride transfer: the redox-active C4(N) atoms are separated by only 3.6 A, and the perceived reaction stereochemistry matches that observed experimentally. The NADH conformation is different in the two dI polypeptides of this form of the dI(2)dIII(1) complex. Comparisons between a number of X-ray structures show that a conformational change in the NADH is driven by relative movement of the two domains which comprise dI. It is suggested that an equivalent conformational change in the intact enzyme is important in gating the hydride-transfer reaction. The observed nucleotide conformational change in the dI(2)dIII(1) complex is accompanied by rearrangements in the orientation of local amino acid side chains which may be responsible for sealing the site from the solvent and polarizing hydride transfer.     

Architecture of complex I and its implications for electron transfer and proton pumping

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H⁺/2e⁻ stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 Å of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.     

Fast hydride transfer in proton-translocating transhydrogenase revealed in a rapid mixing continuous flow device

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).dIII(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).dIII(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.     

The proton pumping mechanism of the bc1 complex

The bc₁ complex is a proton pump of the mitochondrial electron transport chain which transfers electrons from ubiquinol to cytochrome c. It operates via the modified Q cycle in which the two electrons from oxidation of ubiquinol at the Q_o center are bifurcated such that the first electron is passed to Cytc via an iron sulfur center and c₁ whereas the second electron is passed across the membrane by b_L and b_H to reduce ubiquinone at the Q_i center. Proton pumping occurs because oxidation of ubiquinol at the Q_o center releases protons to the P-side and reduction of ubiquinone at the Q_i center takes up protons from the N-side. However, the mechanisms which prevent the thermodynamically more favorable short circuit reactions and so ensure precise bifurcation and proton pumping are not known. Here we use statistical thermodynamics to show that reaction steps that originate from high energy states cannot support high flux even when they have large rate constants. We show how the chemistry of ubiquinol oxidation and the structure of the Q_o site can result in free energy profiles that naturally suppress flux through the short circuit pathways while allowing high rates of bifurcation. These predictions are confirmed through in-silico simulations using a Markov state model.     

Effects of N,N'-dicyclohexylcarbodiimide and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline on hydride ion transfer and proton translocation activities of mitochondrial nicotinamidenucleotide transhydrogenase

N,N'-Dicyclohexylcarbodiimide (DCCD) inhibits the mitochondrial energy-linked nicotinamidenucleotide transhydrogenase (TH). Our studies [Phelps, D.C., & Hatefi, Y. (1981) J. Biol. Chem. 256, 8217-8221; Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480] suggested that the inhibition site of DCCD is near the NAD(H) binding site, because NAD(H) and competitive inhibitors protected TH against inhibition by DCCD and, unlike the unmodified TH, the DCCD-modified TH did not bind to NAD-agarose. Others [Pennington, R.M., & Fisher, R.R. (1981) J. Biol. Chem. 256, 8963-8969] could not demonstrate protection by NADH, obtained data indicating DCCD inhibits proton translocation by TH much more than hydride ion transfer from NADPH to 3-acetylpyridine adenine dinucleotide (AcPyAD), and concluded that DCCD modifies an essential residue in the proton channel of TH. The present studies show that N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) also inhibits TH. The inhibition is pseudo first order at several EEDQ concentrations, and the reaction order with respect to [EEDQ] is unity, suggesting that inhibition involves the interaction of one molecule of EEDQ with one active unit of TH. The EEDQ-modified TH reacts covalently with [3H]aniline, suggesting that the residue modified by EEDQ is a carboxyl group. More significantly, it has been shown that the absorbance change of oxonol VI at 630 minus 603 nm is a reliable reporter of TH-induced membrane potential formation in submitochondrial particles and that TH-catalyzed hydride ion transfer from NADPH to AcPyAD and the membrane potential induced by this reaction are inhibited in parallel by either DCCD or EEDQ.     

The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase

Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase ("dI2dIII1" complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.     

Evidence for a role of a vicinal dithiol in catalysis and proton pumping in mitochondrial nicotinamide nucleotide transhydrogenase

The effect of glutathione, glutathione disulfide and the dithiol reagent phenylarsine oxide on purified soluble as well as reconstituted mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated. Glutathione disulfide and phenylarsine oxide caused an inhibition of transhydrogenase, the extent of which was dependent on the presence of either of the transhydrogenase substrates. In the absence of NADPH glutathione protected partially against inactivation by glutathione disulfide and phenylarsine oxide. In the presence of NADPH glutathione also inhibited transhydrogenase. Reconstituted transhydrogenase vesicles behaved differently as compared to the soluble transhydrogenase and was partially uncoupled by GSSG. It is concluded that transhydrogenase contains a dithiol that is essential for catalysis as well as for proton translocation.     

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.     

Functional dissection of the proton pumping modules of mitochondrial complex I

Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.     

Glutamine 132 in the NAD(H)-binding component of proton-translocating transhydrogenase tethers the nucleotides before hydride transfer

Transhydrogenase, found in bacterial membranes and inner mitochondrial membranes of animal cells, couples the redox reaction between NAD(H) and NADP(H) to proton translocation. In this work, the invariant Gln132 in the NAD(H)-binding component (dI) of the Rhodospirillum rubrum transhydrogenase was substituted with Asn (to give dI.Q132N). Mixtures of the mutant protein and the NADP(H)-binding component (dIII) of the enzyme readily produced an asymmetric complex, (dI.Q132N)(2)dIII(1). The X-ray structure of the complex revealed specific changes in the interaction between bound nicotinamide nucleotides and the protein at the hydride transfer site. The first-order rate constant of the redox reaction between nucleotides bound to (dI.Q132N)(2)dIII(1) was <1% of that for the wild-type complex, and the deuterium isotope effect was significantly decreased. The nucleotide binding properties of the dI component in the complex were asymmetrically affected by the Gln-to-Asn mutation. In intact, membrane-bound transhydrogenase, the substitution completely abolished all catalytic activity. The results suggest that Gln132 in the wild-type enzyme behaves as a "tether" or a "tie" in the mutual positioning of the (dihydro)nicotinamide rings of NAD(H) and NADP(H) for hydride transfer during the conformational changes that are coupled to the translocation of protons across the membrane. This ensures that hydride transfer is properly gated and does not take place in the absence of proton translocation.     

Roles of semiquinone species in proton pumping mechanism by complex I

Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ_Nf) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ_Ns) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ_Nvs) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ_Ns signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.     

Coupling of quinone dynamics to proton pumping in respiratory complex I

Respiratory complex I (NADH:quinone oxidoreductase) plays a central role in generating the proton electrochemical gradient in mitochondrial and bacterial membranes, which is needed to generate ATP. Several high-resolution structures of complex I have been determined, revealing its intricate architecture and complementing the biochemical and biophysical studies. However, the molecular mechanism of long-range coupling between ubiquinone (Q) reduction and proton pumping is not known. Computer simulations have been applied to decipher the dynamics of Q molecule in the ~30 Å long Q tunnel. In this short report, we discuss the binding and dynamics of Q at computationally predicted Q binding sites, many of which are supported by structural data on complex I. We suggest that the binding of Q at these sites is coupled to proton pumping by means of conformational rearrangements in the conserved loops of core subunits.     

A hydride transfer complex reprograms NAD metabolism and bypasses senescence

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Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.     

Essential glycine in the proton channel of Escherichia coli transhydrogenase

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple 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. Previous experiments have established the involvement of three conserved domain II residues in the proton pumping function of the enzyme: His91, Ser139, and Asn222, located on helices 9, 10, and 13, respectively. Eight highly conserved domain II glycines in helices 9, 10, 13, and 14 were mutated to alanine, and the mutant enzymes were assayed for hydride transfer between domains I and III and for proton translocation by domain II. One of the glycines on helix 14, Gly252, was further mutated to Cys, Ser, Thr, and Val, expression levels of the mutant enzymes were evaluated, and each was purified and assayed. The results show that Gly252 is essential for function and support a model for the proton channel composed of helices 9, 10, 13, and 14. Gly252 would allow spatial proximity of His91, Ser139, and Asn222 for proton conductance within the channel. Gly252 mutants are distinguished by high levels of cyclic transhydrogenation activity in the absence of added NADP(H) and by complete loss of proton pumping activity. The purified G252A mutant has <1% proton translocation and reverse transhydrogenation activity, retains 0.9 mol of NADP(H) per domain III, and has 96% intrinsic cyclic transhydrogenation activity, which does not exceed 100% upon the addition of NADP(H). These properties imply that Gly252 mutants exhibit a native-like domain II conformation while blocking proton translocation and coupled exchange of NADP(H) in domain III.     

Protein-protein interactions in pathogen recognition by plants

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitrobinding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies.     

A Drosophila model of mitochondrial disease caused by a complex I mutation that uncouples proton pumping from electron transfer

Mutations affecting mitochondrial complex I, a multi-subunit assembly that couples electron transfer to proton pumping, are the most frequent cause of heritable mitochondrial diseases. However, the mechanisms by which complex I dysfunction results in disease remain unclear. Here, we describe a Drosophila model of complex I deficiency caused by a homoplasmic mutation in the mitochondrial-DNA-encoded NADH dehydrogenase subunit 2 (ND2) gene. We show that ND2 mutants exhibit phenotypes that resemble symptoms of mitochondrial disease, including shortened lifespan, progressive neurodegeneration, diminished neural mitochondrial membrane potential and lower levels of neural ATP. Our biochemical studies of ND2 mutants reveal that complex I is unable to efficiently couple electron transfer to proton pumping. Thus, our study provides evidence that the ND2 subunit participates directly in the proton pumping mechanism of complex I. Together, our findings support the model that diminished respiratory chain activity, and consequent energy deficiency, are responsible for the pathogenesis of complex-I-associated neurodegeneration.KEY WORDS: Mitochondria, Drosophila, Mitochondrial disease, Respiratory chain, Leigh syndrome, Neurodegeneration