Stopped-flow Reaction Kinetics of Recombinant Components of Proton-translocating Transhydrogenase with Physiological Nucleotides

Abstract

Transhydrogenase, which is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria, catalyzes the reaction shown below.A single proton is translocated across the membrane, from the p-aqueous phase (the "outside" of intact mitochondria and bacteria) to the n-aqueous phase (the "inside"), for each hydride equivalent transferred from NADH to NADP ϩ . Under most conditions, this is the in vivo direction; the reaction is driven by the proton electrochemical gradient (⌬p) resulting from the action of respiratory (or photosynthetic) electron transport. For recent reviews, see Refs. 1-3.The gross structure of transhydrogenases from different species is strikingly invariant. The enzyme has three components; dI and dIII, which bind NAD(H) and NADP(H), respectively, protrude from the membrane (on the cytoplasmic side in bacteria, and the matrix side in mitochondria), and dII spans the membrane. Crystal structures of the dIII components of human and bovine transhydrogenases were recently described (4 -6), and the solution structure of the Rhodospirillum rubrum equivalent was determined by NMR.1 The basic fold of dIII is similar to the classical, dinucleotide-binding domain of lactate dehydrogenase, but NADP ϩ is bound with an unusual, "reversed" orientation. The nicotinamide ring of the bound NADP ϩ is exposed on a ridge of dIII. A homology model of dI (8), based on the crystal structure of the sequence-related alanine dehydrogenase (9), suggests that the nicotinamide ring of NADH is located in a deep cleft. It was proposed that, in the complete transhydrogenase, the ridge of dIII inserts into the cleft of dI to bring the nicotinamide rings of the two nucleotides into apposition to effect direct hydride transfer (5, 6). The protruding helix-D/loop-D of dIII is thought to interact with the membrane-spanning, dII and, together with the adjacent, lidlike loop E, which passes over the bound nucleotide, might be responsible for the energy transmission. The proton translocation steps during turnover are probably coupled specifically to changes in the mode of NADP(H) binding (1,5,10).Kinetic studies of transhydrogenase have also developed, following discoveries that fragments of the protein retain their nucleotide binding and some catalytic capacity. Thus, recombinant dI and dIII proteins, which bind their cognate nucleotides, have now been isolated from a number of species (11-16); mixtures of these proteins, even from different species, catalyze transhydrogenation reactions, albeit with properties that are modified relative to those of the complete enzyme. Transient state experiments, in particular, have revealed useful information on the hydride transfer step (17)(18)(19). Without exception, experiments with dI⅐dIII complexes have been carried out with nucleotide analogues having altered absorbance spectra to facilitate measurement of the rate of reaction. In this report we describe transient state experiments with the physiological nucleotides. A procedure is described, in which singl...

Crystal Structures of Transhydrogenase Domain I with and without Bound NADH,

“…Motion of domain III within TH is described in terms of 'open' and 'occluded' states in the 'alternating site, binding change mechanism' (11). Based on kinetic data for the heterotrimeric complex in solution (24,31), the cocrystal structure with NADP bound to domain III, e.g., Figure 7, corresponds to the 'occluded' state (11). Recently, a conserved acidic residue ( Asp213) in an extramembranous loop of domain II of E. coli TH has been found to be required for proton translocation, suggesting a mechanism by which NADPH-induced movement of domain III could affect a pK a shift in domain II, which in turn would favor proton translocation (36).…”

“…Kinetic experiments have defined two states for bound NADH, one with a K d of ∼30 µM, and one with a K d of ∼300 µM (24,25). These binding constants are interpreted as corresponding to favorable binding when domain I is associated with domain III (with NADP bound) in the 'open' state, and weaker binding when domain III is converted to the 'occluded' state prior to rapid hydride transfer (11).…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…Thus, the basic mechanism is (i) substrate nucleotides, NADP + and NADH, associate with their binding sites in an enzyme protomer having both dIII and dI in open states, (ii) protonation of dII from the outside aqueous phase converts the open dIII to occluded, (iii) hydride-ion equivalents are transferred from bound NADH to NADP + , (iv) deprotonation of dII on the cytoplasmic side regenerates the open state of dIII, and (v) the nucleotide products, NADPH and NAD + , are released. The binding affinities of NAD + and NADH to dI are not significantly altered during the catalytic cycle; this enzyme component remains open except perhaps during the brief (<10 À3 s) period of hydride transfer [23,56]. The proposed mechanism affords two important features.…”

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

“…Following experiments to characterize the hydride transfer reaction between NADPH and NAD ϩ in mixtures of dI and dIII, we showed that the value of the rate constant for product NADH release, measured using isolated dI, was inconsistent with the simple kinetic scheme (34). It was suggested that the rate constant for NADH release from the complex of dI and dIII must be much greater than that for isolated dI protein.…”

“…However, assuming that the binding capacity of the low affinity site is 1.0/mol of dI 2 dIII 1 , the data fitted to a K d ϭ 311 Ϯ 91 M and an enthalpy change of Ϫ79.4 kJ⅐mol Ϫ1 of dI 2 dIII 1 . In recent stopped-flow experiments, we recorded the kinetics of the fluorescence quenching of the (single) Trp 72 in dI during NADH binding (34); the objective was to determine the k on and k off for that nucleotide-binding step. We noted that the amplitude of the dI fluorescence quenching was decreased when dIII.NADPH was present (the wild-type dIII protein, with no Trp residues, was used).…”