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.     

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.     

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.     

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.     

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.     

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     

Protein-protein recognition and the association of immunoglobulin constant domains

Four immunoglobulin constant domain interfaces were analysed to determine how the light and heavy chains of the domains recognize each other and secondarily, why the packing of the beta sheets is different from that normally observed in the interior of domains of beta sheet proteins. It was found that two bulky groups in the centre of each of the domain interfaces prevent the alignment of sheet axes by intercalating with residues in the sheet opposite. This intercalation forms the basis of constant domain light chain-heavy chain recognition.     

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O(2) to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to Cu(A) in CcO with a rate constant of 60,000s(-1). Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into Cu(A) of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.     

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(+)/2e indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(+)/2e . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.     

The Rhodospirillum rubrum cytochrome bc1 complex: redox properties, inhibitor sensitivity and proton pumping.

A detergent-solubilized, three-subunit-containing cytochrome bc1 complex, isolated from the photosynthetic bacterium R. rubrum, has been shown to be highly sensitive to stigmatellin, myxothiazol, antimycin A and UHDBT, four specific inhibitors of these complexes. Oxidation-reduction titrations have allowed the determination of Em values for all the electron-carrying prosthetic groups in the complex. Antimycin A has been shown to produce a red shift in the alpha-band absorbance maximum of one of the cytochrome b hemes in the complex and stigmatellin has been shown to alter both the Em and EPR g-values of the Rieske iron-sulfur protein in the complex. Western blots have revealed antigenic similarities between the cytochrome subunits of the R. rubrum complex and those of the related photosynthetic bacteria, Rb. capsulatus and Rb. sphaeroides. The R. rubrum complex has been incorporated into liposomes. These liposomes exhibit respiratory control and are able to couple electron transfer from quinol to cytochrome c to proton translocation across the liposome membrane in a manner consistent with a Q-cycle mechanism. It can thus be concluded that neither electron transport nor coupled proton translocation by the cytochrome bc1 complex requires more than three subunits in R. rubrum.     

Electron transfer and proton pumping under excitation of dark-adapted chloroplasts with flashes of light

Proton release inside thylakoids, which is linked to the action of the water-oxidizing enzyme system, was investigated spectrophotometrically with the dye neutral red under conditions when the external phase was buffered. Under excitation of dark-adapted chloroplasts with four short laser flashes in series, the pattern of proton release as a function of the flash number was recorded and interpreted in the light of the generally accepted scheme for consecutive transitions of the water-oxidizing enzyme system: S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄, S₀. It was found that the proton yield after the first flash varied in a reproducible manner, being dependent upon the dark pretreatment given. In terms of the proton-electron reaction during these transitions, the pattern was as follows. In strictly dark-adapted chloroplasts (frozen chloroplasts thawed in darkness and kept for at least 7 min in the dark after dilution), it was fitted well by a stoichiometry of 1:0:1:2. In a less stringently dark-adapted preparation (as above but thawed under light), it was fitted by 0:1:1:2. Mechanistically this is not yet understood. However, it is a first step towards resolving controversy over this pattern among different laboratories. Under conditions where the 1:0:1:2 stoichiometry was observed, proton release was time resolved. Components with half-rise times of 500 and 1000 μs could be correlated with the S₂ → S₃ and S₃ → S₄ transitions, respectively. Proton release during the S₀ → S₁ transition is more rapid, but is less well attributable to the transitions due to error proliferation. A distinct component with a half-rise time of only 100 μs was observed after the second flash. Since it did not fit into the expected kinetics (based on literature data) for the S_i → S_i+1 transitions, we propose that it reflects proton release from a site which is closer to the reaction center of Photosystem (PS) II than the water-splitting enzyme system. This is supported by the observation of rapid proton release under conditions where water oxidation is blocked. Related experiments on the pattern of proton uptake at the reducing side of PS II indicated that protons act as specific counterions for semiquinone anions without binding to them.