The crystal structure of an asymmetric complex of the two nucleotide binding components of proton-translocating transhydrogenase

BACKGROUND: Membrane-bound ion translocators have important functions in biology, but their mechanisms of action are often poorly understood. Transhydrogenase, found in animal mitochondria and bacteria, links the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. Linkage is achieved through changes in protein conformation at the nucleotide binding sites. The redox reaction takes place between two protein components located on the membrane surface: dI, which binds NAD(H), and dIII, which binds NADP(H). A third component, dII, provides a proton channel through the membrane. Intact membrane-located transhydrogenase is probably a dimer (two copies each of dI, dII, and dIII). RESULTS: We have solved the high-resolution crystal structure of a dI:dIII complex of transhydrogenase from Rhodospirillum rubrum-the first from a transhydrogenase of any species. It is a heterotrimer, having two polypeptides of dI and one of dIII. The dI polypeptides fold into a dimer. The loop on dIII, which binds the nicotinamide ring of NADP(H), is inserted into the NAD(H) binding cleft of one of the dI polypeptides. The cleft of the other dI is not occupied by a corresponding dIII component. CONCLUSIONS: The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase: effect of substrates on the sensitivity of the enzyme to trypsin and identification of tryptic cleavage sites

The mitochondrial nicotinamide nucleotide transhydrogenase catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner mitochondrial membrane. The enzyme (1043 residues) is composed of an N-terminal hydrophilic segment (approximately 400 residues long) which binds NAD(H), a C-terminal hydrophilic segment (approximately 200 residues long) which binds NADP(H), and a central hydrophobic segment (approximately 400 residues long) which appears to form about 14 membrane-intercalating clusters of approximately 20 residues each. Substrate modulation of transhydrogenase conformation appears to be intimately associated with its mechanism of proton translocation. Using trypsin as a probe of enzyme conformation change, we have shown that NADPH (and to a much lesser extent NADP) binding alters transhydrogenase conformation, resulting in increased susceptibility of several bonds to tryptic hydrolysis. NADH and NAD had little or no effect, and the NADPH concentration for half-maximal enhancement of trypsin sensitivity of transhydrogenase activity (35 microM) was close to the Km of the enzyme for NADPH. The NADPH-promoted trypsin cleavage sites were located 200-400 residues distant from the NADP(H) binding domain near the C-terminus. For example, NADPH binding greatly increased the trypsin sensitivity of the K410-T411 bond, which is separated from the NADP(H) binding domain by the 400-residue-long membrane-intercalating segment. It also enhanced the tryptic cleavage of the R602-L603 bond, which is located within the central hydrophobic segment. These results, which suggest a protein conformation change as a result of NADPH binding, have been discussed in relation to the mechanism of proton translocation by the transhydrogenase.     

Structure and mechanism of proton-translocating transhydrogenase

Recent developments have led to advances in our understanding of the structure and mechanism of action of proton-translocating (or AB) transhydrogenase. There is (a) a high-resolution crystal structure, and an NMR structure, of the NADP(H)-binding component (dIII), (b) a homology-based model of the NAD(H)-binding component (dI) and (c) an emerging consensus on the position of the transmembrane helices (in dII). The crystal structure of dIII, in particular, provides new insights into the mechanism by which the energy released in proton translocation across the membrane is coupled to changes in the binding affinities of NADP(+) and NADPH that drive the chemical reaction.     

A hybrid of the transhydrogenases from Rhodospirillum rubrum and Mycobacterium tuberculosis catalyses rapid hydride transfer but not the complete, proton-translocating reaction

All transhydrogenases appear to have three components: dI, which binds NAD(H), and dIII, which binds NADP(H), protrude from the membrane, and dII spans the membrane. However, the polypeptide composition of the enzymes varies amongst species. The transhydrogenases of Mycobacterium tuberculosis and of Rhodospirillum rubrum have three polypeptides. Sequence analysis indicates that an ancestral three-polypeptide enzyme evolved into transhydrogenases with either two polypeptides (such as the Escherichia coli enzyme) or one polypeptide (such as the mitochondrial enzyme). The fusion steps in each case probably led to the development of an additional transmembrane helix. A hybrid transhydrogenase was constructed from the dI component of the M. tuberculosis enzyme and the dII and dIII components of the R. rubrum enzyme. The hybrid catalyses cyclic transhydrogenation but not the proton-translocating, reverse reaction. This shows that nucleotide-binding/release at the NAD(H) site, and hydride transfer, are fully functional but that events associated with NADP(H) binding/release are compromised. It is concluded that sequence mismatch in the hybrid prevents a conformational change between dI and dIII which is essential for the step accompanying proton translocation.     

The membrane-peripheral subunits of transhydrogenase from Entamoeba histolytica are functional only when dimerized

Unlike their bacterial and mammalian counterparts, the NADP(H)- and NAD(H)-binding components of proton-translocating transhydrogenase from the protozoan parasite Entamoeba histolytica (denoted ehdIII and ehdI, respectively) are tethered by a polypeptide linker. The recombinant tethered fragment, ehdIII-ehdI, was prepared without its membrane-spanning dII component. Dimers of ehdIII-ehdI catalyzed transhydrogenation, but monomers were inactive. The addition of ehdIII to ehdIII-ehdI monomers did not lead to an increase in the rate of transhydrogenation, showing that this inactivity is not the result of an unfavorable topology introduced by the linker. The addition of a bacterial dI to ehdIII-ehdI led to an increase in the rate of transhydrogenation, showing that the linker is flexible. A hybrid protein in which ehdIII is tethered to the bacterial dI (denoted ehdIII-rrdI) more readily formed active dimers. Data from small angle x-ray scattering by the hybrid dimers were fitted to models derived from the high-resolution crystal structure of the bacterial dI(2)dIII(1) complex (Cotton, N. P. J., White, S. A., Peake, S. J., McSweeney, S., and Jackson, J. B. (2001) Structure 9, 165-T176). The results show that the ehdIII-rrdI dimer is asymmetric; one dIII associates with dI, as in the bacterial complex, but the other is displaced. The results provide evidence for the alternating site, binding change model for proton translocation by intact transhydrogenase.     

A hybrid of the transhydrogenases from Rhodospirillum rubrum and Mycobacterium tuberculosis catalyses rapid hydride transfer but not the complete, proton-translocating reaction

All transhydrogenases appear to have three components: dI, which binds NAD(H), and dIII, which binds NADP(H), protrude from the membrane, and dII spans the membrane. However, the polypeptide composition of the enzymes varies amongst species. The transhydrogenases of Mycobacterium tuberculosis and of Rhodospirillum rubrum have three polypeptides. Sequence analysis indicates that an ancestral three-polypeptide enzyme evolved into transhydrogenases with either two polypeptides (such as the Escherichia coli enzyme) or one polypeptide (such as the mitochondrial enzyme). The fusion steps in each case probably led to the development of an additional transmembrane helix. A hybrid transhydrogenase was constructed from the dI component of the M. tuberculosis enzyme and the dII and dIII components of the R. rubrum enzyme. The hybrid catalyses cyclic transhydrogenation but not the proton-translocating, reverse reaction. This shows that nucleotide-binding/release at the NAD(H) site, and hydride transfer, are fully functional but that events associated with NADP(H) binding/release are compromised. It is concluded that sequence mismatch in the hybrid prevents a conformational change between dI and dIII which is essential for the step accompanying proton translocation.     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K(d) values) for NADPH (0.87 microM), NADP(+) (16 microM), NADH (50 microM), and NAD(+) (100-500 microM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K(d) values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K(d) values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidized.     

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.     

Interactions of the NADP(H)-binding domain III of proton-translocating transhydrogenase from escherichia coli with NADP(H) and the NAD(H)-binding domain I studied by NMR and site-directed mutagenesis

Using the purified NADP(H)-binding domain of proton-translocating Escherichia coli transhydrogenase (ecIII) overexpressed in (15)N- and (2)H-labeled medium, together with the purified NAD(H)-binding domain from E. coli (ecI), the interface between ecIII and ecI, the NADP(H)-binding site and the influence on the interface by NAD(P)(H) was investigated in solution by NMR chemical shift mapping. Mapping of the NADP(H)-binding site showed that the NADP(H) substrate is bound to ecIII in an extended conformation at the C-terminal end of the parallel beta-sheet. The distribution of chemical shift perturbations in the NADP(H)-binding site, and the nature of the interaction between ecI and ecIII, indicated that the nicotinamide moiety of NADP(H) is located near the loop comprising residues P346-G353, in agreement with the recently determined crystal structures of bovine [Prasad, G. S., et al. (1999) Nat. Struct. Biol. 6, 1126-1131] and human heart [White, A. W., et al. (2000) Structure 8, 1-12] transhydrogenases. Further chemical shift perturbation analysis also identified regions comprising residues G389-I406 and G430-V434 at the C-terminal end of ecIII's beta-sheet as part of the ecI-ecIII interface, which were regulated by the redox state of the NAD(P)(H) substrates. To investigate the role of these loop regions in the interaction with domain I, the single cysteine mutants T393C, R425C, G430C, and A432C were generated in ecIII and the transhydrogenase activities of the resulting mutant proteins characterized using the NAD(H)-binding domain I from Rhodospirillum rubrum (rrI). All mutants except R425C showed altered NADP(H) binding and domain interaction properties. In contrast, the R425C mutant showed almost exclusively changes in the NADP(H)-binding properties, without changing the affinity for rrI. Finally, by combining the above conclusions with information obtained by a further characterization of previously constructed mutants, the implications of the findings were considered in a mechanistic context.     

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.     

Structural changes in the recombinant, NADP(H)-binding component of proton translocating transhydrogenase revealed by NMR spectroscopy

We have analysed 1H, 15N-HSQC spectra of the recombinant, NADP(H)-binding component of transhydrogenase in the context of the emerging three dimensional structure of the protein. Chemical shift perturbations of amino acid residues following replacement of NADP+ with NADPH were observed in both the adenosine and nicotinamide parts of the dinucleotide binding site and in a region which straddles the protein. These observations reflect the structural changes resulting from hydride transfer. The interactions between the recombinant, NADP(H)-binding component and its partner, NAD(H)-binding protein, are complicated. Helix B of the recombinant, NADP(H)-binding component may play an important role in the binding process.     

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.     

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)     

The proton-translocating nicotinamide–dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an -->H(+)/2e(-) quotient of 1.94+/-0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K(+)-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of -450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.     

The interaction of mitochondrial transhydrogenase with derivatives of coenzyme A

The 2,4-dinitrophenyl derivative of dephospho-CoA and the 7-nitrobenzofurazan-4-yl derivative of CoA are competitive inhibitors (Ki 3 microM and 2.6 microM respectively) of mitochondrial transhydrogenase with regard to NAD+ and NADPH respectively. The 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA is a competitive inhibitor with regard to both transhydrogenase substrates with the same Ki equal to 0.3 microM. The pattern of transhydrogenase inhibition with the 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA indicates that one molecule of the inhibitor binds simultaneously to both the NADP(H) and the NAD(H) binding sites of the enzyme. This result is evidence of the short distance between the NADP(H) and the NAD(H) binding sites.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

Zinc ions selectively inhibit steps associated with binding and release of NADP(H) during turnover of proton-translocating transhydrogenase

Transhydrogenase couples the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. In membrane vesicles from Escherichia coli and Rhodospirillum rubrum, the transhydrogenase reaction (measured in the direction driving inward proton translocation) was inhibited by Zn(2+) and Cd(2+). However, depending on pH, the metal ions either had no effect on, or stimulated, "cyclic" transhydrogenation. They must, therefore, interfere specifically with steps involving binding/release of NADP(+)/NADPH: the steps thought to be associated with proton translocation. It is suggested that Zn(2+) and Cd(2+) bind in the proton-transfer pathway and block inter-conversion of states responsible for changing NADP(+)/NADPH binding energy.     

X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli

The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.     

Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.     

Midgut mitochondrial transhydrogenase in wandering stage larvae of the tobacco hornworm, Manduca sexta

Midgut mitochondria from fifth larval instar Manduca sexta exhibited a transhydrogenase that catalyzes the following reversible reaction: NADPH + NAD(+) <--> NADP(+) + NADH. The NADPH-forming transhydrogenation occurred as a nonenergy- and energy-linked activity. Energy for the latter was derived from the electron transport-dependent utilization of NADH or succinate, or from Mg++-dependent ATP hydrolysis by ATPase. The NADH-forming and all of the NADPH-forming reactions appeared optimal at pH 7.5, were stable to prolonged dialysis, and displayed thermal lability. N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the NADPH --> NAD(+) and energy-linked NADH --> NADP(+) transhydrogenations, but not the nonenergy-linked NADH --> NADP(+) reaction. Oligomycin only inhibited the ATP-dependent energy-linked activity. The NADH-forming, nonenergy-linked NADPH-forming, and the energy-linked NADPH-forming activities were membrane-associated in M. sexta mitochondria. This is the first demonstration of the reversibility of the M. sexta mitochondrial transhydrogenase and, more importantly, the occurrence of nonenergy-linked and energy-linked NADH --> NADP(+) transhydrogenations. The potential relationship of the transhydrogenase to the mitochondrial, NADPH-utilizing ecdysone-20 monooxygenase of M. sexta is considered.