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 heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation.

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 nucleotide-binding 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.

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

Transhydrogenase couples the transfer of hydride-ion equivalents between NAD(H) and NADP(H) to proton translocation across a membrane. The enzyme has three components: dI binds NAD(H), dIII binds NADP(H) and dII spans the membrane. Coupling between transhydrogenation and proton translocation involves changes in the binding of NADP(H). Mixtures of isolated dI and dIII from Rhodospirillum rubrum transhydrogenase catalyse a rapid, single-turnover burst of hydride transfer between bound nucleotides; subsequent turnover is limited by NADP(H) release. Stopped-flow experiments showed that the rate of the hydride transfer step is decreased at low pH. Single Trp residues were introduced into dIII by site-directed mutagenesis. Two mutants with similar catalytic properties to those of the wild-type protein were selected for a study of nucleotide release. The way in which Trp fluorescence was affected by nucleotide occupancy of dIII was different in the two mutants, and hence two different procedures for determining the rate of nucleotide release were developed. The apparent first-order rate constants for NADP(+) release and NADPH release from isolated dIII increased dramatically at low pH. It is concluded that a single ionisable group in dIII controls both the rate of hydride transfer and the rate of nucleotide release. The properties of the protonated and unprotonated forms of dIII are consistent with those expected of intermediates in the NADP(H)-binding-change mechanism. The ionisable group might be a component of the proton-translocation pathway in the complete enzyme.

Stopped-flow reaction kinetics of recombinant components of proton-translocating transhydrogenase with physiological nucleotides.

New information on the high resolution structure of the membrane proton pump, transhydrogenase, now provides a framework for understanding kinetic descriptions of the enzyme. Here, we have studied redox reactions catalyzed by mixtures of the recombinant NAD(H)-binding component (dI) of Rhodospirillum rubrum transhydrogenase, and the recombinant NADP(H)-binding component (dIII) of either the R. rubrum enzyme or the human enzyme. By recording changes in the fluorescence emission of native and engineered Trp residues, the rates of the redox reaction with physiological nucleotides have been measured under stopped-flow conditions, for the first time. Rate constants for the binding reaction between NAD(+)/NADH and the R. rubrum dI.dIII complex are much greater than those between nucleotide and isolated dI. For the redox step between the physiological nucleotides on the R. rubrum dI. dIII complex, the rate constant in the forward direction, k(f) approximately 2900 s(-1), and that for the reverse reaction, k(r) approximately 110 s(-1). Comparisons with reactions involving an analogue of NAD(H) indicate that the rate constants at this step are strongly affected by the redox driving force.

Evidence for the stabilization of NADPH relative to NADP(+) on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence.

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria (hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase (rrdIII.D155W), are more fluorescent when NADP(+) is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP(+)/NADPH bound to the dIII proteins is some 60-70 mV higher than that in free solution. This results in favoured reduction of NADP(+) by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.

A shift in the equilibrium constant at the catalytic site of proton-translocating transhydrogenase: significance for a 'binding-change' mechanism.

In mitochondria and bacteria, transhydrogenase uses the transmembrane proton gradient (Deltap) to drive reduction of NADP+ by NADH. We have investigated the pre-steady-state kinetics of NADP+ reduction by acetylpyridine adenine dinucleotide (AcPdADH, an analogue of NADH) in complexes formed from the two, separately prepared, recombinant, peripheral subunits of the enzyme: the dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH. In the stopped-flow spectrophotometer the reaction proceeds as a single-turnover burst of hydride transfer to NADP+ on dIII before product NADPH release becomes limiting in steady state. The burst is biphasic. The results indicate that the fast phase represents direct hydride transfer from AcPdADH to NADP+ in dI:dIII complexes, and that the slow phase, which predominates when [dI]<[dIII], corresponds to dissociation of the protein complexes during multiple turnovers of dI. Measurements on the amplitude of the burst, and on the apparent first-order rate constant of the fast phase, indicate that the equilibrium constant of the hydride-transfer step on the enzyme is shifted relative to that in solution. This has consequences for a model proposed earlier, in which Deltap is used, not at the hydride-transfer step, but to change the binding affinities of NADP+ and NADPH.

A catalytically active complex formed from the recombinant dI protein of Rhodospirillum rubrum transhydrogenase, and the recombinant dIII protein of the human enzyme.

Transhydrogenase is a proton pump. It has three components: dI and dIII protrude from the membrane and contain the binding sites for NAD(H) and NADP(H), respectively, and dII spans the membrane. We have expressed dIII from Homo sapiens transhydrogenase (hsdIII) in Escherichia coli. The purified protein was associated with stoichiometric amounts of NADP(H) bound to the catalytic site. The NADP+ and NADPH were released only slowly from the protein, supporting the suggestion that nucleotide-binding by dIII is regulated by the membrane-spanning dII. HsdIII formed a catalytically active complex with recombinant dI from Rhodospirillum rubrum (rrdI), even in the absence of dII. The rates of forward and reverse transhydrogenation catalysed by this complex are probably limited by slow release from dIII of NADPH and NADP+, respectively. The hybrid complex also catalysed high rates of 'cyclic' transhydrogenation, indicating that hydride transfer, and exchange of nucleotides with dI, are rapid. Stopped-flow experiments revealed a rapid, monoexponential, single-turnover burst of reverse transhydrogenation in pre-steady-state. The apparent first-order rate constant of the burst increased with the concentration of rrdI. A deuterium isotope effect (kH/kD approximately 2 at 27 degrees C) was observed when [4B-1H]NADPH was replaced with [4B-2H]NADPH. The characteristics of the burst of transhydrogenation with rrdI:hsdIII differed from those previously reported for rrdI:rrdIII (J.D. Venning et al., Eur. J. Biochem. 257 (1998) 202-209), but the differences are readily explained by a greater dissociation constant of the hybrid complex. The steady-state rate of reverse transhydrogenation by the rrdI:hsdIII complex was almost independent of pH, but there was a single apparent pKa ( approximately 9.1) associated with the cyclic reaction. The reactions of the dI:dIII complex probably proceed independently of those protonation/deprotonation reactions which, in the complete enzyme, are associated with H+ translocation.

Catalytic properties of hybrid complexes of the NAD(H)-binding and NADP(H)-binding domains of the proton-translocating transhydrogenases from Escherichia coli and Rhodospirillum rubrum.

Transhydrogenase couples reversible hydride transfer from NADH to NADP+ to proton translocation across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. The enzyme is composed of three parts. Domain I (dI) and domain III (dIII) are water soluble and contain the binding sites for NAD(H) and NADP(H), respectively; domain II (dII) spans the membrane. In the present investigation, dI from Rhodospirillum rubrum (rrI) and Escherichia coli (ecI), and dIII from R. rubrum (rrIII) and E. coli (ecIII) were overexpressed in E. coli and subsequently purified. Also, a preparation of a partially degraded E. coli transhydrogenase (ecbeta) was examined. Catalytic activities were analyzed in various dI+dIII and dI+ecbeta combinations. The abilities of the different dI+dIII combinations to catalyze cyclic transhydrogenation, i.e., the reduction of AcPyAD+ by NADH mediated via tightly bound NADP(H) in dIII, varied in the order: rrI+ecIII approximately rrI+rrIII > rrI+ecbeta >> ecI+ecIII; no measurable activities for ecI+rrIII and ecI+ecbeta were detected. Thus, rrI has a much greater apparent affinity than ecI for ecIII or rrIII or ecbeta. The pH dependences of the cyclic reaction seem to be determined by scalar protonation events on dI, both in rrI+rrIII and ecI+ecIII mixtures as well as in the wild-type R. rubrum and possibly in the E. coli enzyme. Higher reverse activities for rrI+ecbeta than for rrI+ecIII confirmed the regulatory role of dII for the association and dissociation rates of NADP(H).

Stopped-flow kinetics of hydride transfer between nucleotides by recombinant domains of proton-translocating transhydrogenase.

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD+/NADH, and domain III for NADP+/NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP+ (k approximately 0.03 s(-1)). Phase A of the burst (k approximately 600 s(-1)) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (k approximately 10-50 s(-1)), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or release. A comparison of the temperature dependences of AcPdAD+ reduction by [4B-2H]NADPH, and by [4B-1H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272, 27535-27538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I:III complex.

Mutation of amino acid residues in the mobile loop region of the NAD(H)-binding domain of proton-translocating transhydrogenase.

The effects of single amino acid substitutions in the mobile loop region of the recombinant NAD(H)-binding domain (dI) of transhydrogenase have been examined. The mutations lead to clear assignments of well-defined resonances in one-dimensional 1H-NMR spectra. As with the wild-type protein, addition of NADH, or higher concentrations of NAD+, led to broadening and some shifting of the well-defined resonances. With many of the mutant dI proteins more nucleotide was required for these effects than with wild-type protein. Binding constants of the mutant proteins for NADH were determined by equilibrium dialysis and, where possible, by NMR. Generally, amino acid changes in the mobile loop region gave rise to a 2-4-fold increase in the dI-nucleotide dissociation constants, but substitution of Ala236 for Gly had a 10-fold effect. The mutant dI proteins were reconstituted with dI-depleted bacterial membranes with apparent docking affinities that were indistinguishable from that of wild-type protein. In the reconstituted system, most of the mutants were more inhibited in their capacity to perform cyclic transhydrogenation (reduction of acetyl pyridine adenine dinucleotide, AcPdAD+, by NADH in the presence of NADP+) than in either the simple reduction of AcPdAD+ by NADPH, or the light-driven reduction of thio-NADP+ by NADH, which suggests that they are impaired at the hydride transfer step. A cross-peak in the 1H-1H nuclear Overhauser enhancement spectrum of a mixture of wild-type dI and NADH was assigned to an interaction between the A8 proton of the nucleotide and the betaCH3 protons of Ala236. It is proposed that, following nucleotide binding, the mobile loop folds down on to the surface of the dI protein, and that contacts, especially from Tyr235 in a Gly-Tyr-Ala motif with the adenosine moiety of the nucleotide, set the position of the nicotinamide ring of NADH close to that of NADP+ in dIII to effect direct hydride transfer.

Interdomain hydride transfer in proton-translocating transhydrogenase.

We describe the use of the recombinant, nucleotide-binding domains (domains I and III) of transhydrogenase to study structural, functional and dynamic features of the protein that are important in hydride transfer and proton translocation. Experiments on the transient state kinetics of the reaction show that hydride transfer takes place extremely rapidly in the recombinant domain I:III complex, even in the absence of the membrane-spanning domain II. We develop the view that proton translocation through domain II is coupled to changes in the binding characteristics of NADP+ and NADPH in domain III. A mobile loop region which emanates from the surface of domain I, and which interacts with NAD+ and NADH during nucleotide binding has been studied by NMR spectroscopy and site-directed mutagenesis. An important role for the loop region in the process of hydride transfer is revealed.

The pH dependences of reactions catalyzed by the complete proton-translocating transhydrogenase from Rhodospirillum rubrum, and by the complex formed from its recombinant nucleotide-binding domains.

Transhydrogenase couples the translocation of protons across a membrane to the transfer of reducing equivalents between NAD(H) and NADP(H). Using transhydrogenase from Rhodospirillum rubrum we have examined the pH dependences of the 'forward' and 'reverse' reactions, and of the 'cyclic' reaction (NADP(H)-dependent reduction of the analogue, acetyl pyridine adenine dinucleotide, by NADH). In the case of the membrane-bound protein in chromatophores, the imposition of a protonmotive force through the action of the light-driven electron-transport system, stimulated forward transhydrogenation, inhibited reverse transhydrogenation, but had no effect on the cyclic reaction. The differential response at a range of pH values provides evidence that hydride transfer per se is not coupled to proton translocation and supports the view that energy transduction occurs at the level of NADP(H) binding. Chromatophore transhydrogenase and the detergent-dispersed enzyme both have bell-shaped pH dependences for forward and reverse transhydrogenation. The cyclic reaction, however, is rapid at low and neutral pH, and is attenuated only at high pH. A mixture of recombinant purified NAD(H)-binding domain I, and NADP(H)-binding domain III, of R. rubrum transhydrogenase carry out the cyclic reaction with a similar pH profile to that of the complete enzyme, but the forward and reverse reactions were much less pH dependent. The rates of release of NADP+ and of NADPH from isolated domain III were pH independent. The results are consistent with a model for transhydrogenation, in which proton binding from one side of the membrane is consequent upon the binding of NADP+ to the enzyme, and then proton release on the other side of the membrane precedes NADPH release.

Evidence that the transfer of hydride ion equivalents between nucleotides by proton-translocating transhydrogenase is direct.

The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+. Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation.

Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli.

The effect of each of 20 different amino acid supplements to the growth medium of Escherichia coli on the extracellular release of a periplasmic recombinant cytochrome b5 was investigated. Only glycine, and to a lesser extent histidine, stimulated the synthesis of secretory cytochrome b5, as well as its discharge into the medium. Extracellular amounts of cytochrome b5 accrued with increasing concentrations of exogenous glycine and duration of the culture period, in spite of the fact that increasing glycine in the medium progressively inhibited cell growth. For example, 1% medium glycine caused a 50% reduction in bacterial growth, but doubled the periplasmic pool of cytochrome b5 to over 25 micrograms of cytochrome b5/ml of culture at 24 h, a period during which almost all of cellular haemoprotein pool was turned over into the medium. A comparative study of the exportable form of cytochrome b5 with a (non-secretory) cytoplasmic-resident counterpart indicated that the periplasmic cytochrome b5 content was selectively discharged into the medium when less than 1% glycine was present, but, at higher doses, a significant proportion of the additional extracellular haemoprotein was derived from cell lysis. Optimal level of periplasmic discharge of the cytochrome required both active protein synthesis and the presence of a glycine supplement in the medium from the onset of bacterial growth. Phase-contrast and scanning electron microsocopy of glycine-grown Escherichia coli showed that the cells had a 3-7-fold enlarged "eyeball' spheroidal morphology, with a condensed pericircular cytoplasm. The bulk of the volume in such hypertrophied cells consisted of the periplasm; this was reflected by the progressively lowered buoyancy of E. coli cultured with increasing amounts of glycine. The fragility of such cells was apparent by their marked sensitivity to lysis at glycine concentrations above 1%. We conclude that supplementation of E. coli cultures with moderate amounts of glycine substantially stimulates the synthesis of exportable proteins and further enhances their yield by discharge into the growth medium.