Structural basis for the light regulation of chloroplast NADP malate dehydrogenase

The activity of chloroplast NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) in both C3 and C4 plants is regulated by light intensity. In darkness, the activity of the enzyme can be less than 1% of the maximal activity found at high light intensities. The extent of activation in the light is dynamic, responding rapidly to changes in light intensity and adapting to changes in photosynthetic rate. Enzyme activation is caused by thioredoxin-catalyzed reduction of two regulatory disulfide bonds, while inactivation is accomplished by thioredoxin-catalyzed re-oxidation. In the case of NADP-MDH, the coenzyme substrates NADP+ and NADPH modify the rate of this interconversion and seem to be important to the extent of activation in vivo. The recent determination of the X-ray structure of the oxidized, dark form of NADP-MDH from the C4 plants Flaveria bidentis and Sorghum shows how oxidation of a disulfide bond can inactivate the enzyme. This review discusses the various structural features of NADP-MDH that seem to be responsible for the regulatory properties of the enzyme and emphasizes that large changes of activity can be accomplished by multiple, small, reinforcing changes rather than a single large change in a signal molecule concentration.     

Structural and catalytic properties of oxidized and reduced chloroplast NADP-malate dehydrogenase upon denaturation and renaturation

Chloroplast NADP-dependent malate dehydrogenase exists in two interconvertible forms: the inactive disulfide-containing form and the active dithiol form. No major difference in secondary structure or conformation was found between the oxidized and the reduced enzyme as determined by circular dichroism and intrinsic protein fluorescence. The guanidine/HCl-dependent unfolding of the enzyme is characterized by two transition midpoints: those of the reduced enzyme are lower by about 0.2 M guanidine/HCl compared to the oxidized enzyme. As shown by analytical ultracentrifugation, there was no effect of guanidine/HCl concentrations up to 0.25 M on the quaternary structure of the enzyme in its oxidized and reduced forms: both sedimentation coefficient (S20,w = 4.9 +/- 0.1 S) and sedimentation equilibrium (75 +/- 3 kDa) yield the dimer. In the oxidized state the enzyme undergoes guanidine-dependent dissociation to the monomer with a midpoint of transition at 0.5 M. The kinetics of unfolding were found to be significantly faster for the reduced than for the oxidized enzyme. Renaturation and reactivation of reduced enzyme was more rapid and occurred with higher yields (100%) than for the oxidized enzyme (60-80% yield). Furthermore, the effect of denaturants on catalytic activity, and reductive activation of the oxidized form, were studied. Both increase in protein fluorescence and a stimulatory effect on the activities at low guanidine/HCl concentrations were observed for the oxidized and the reduced form of the enzyme. Denaturants increase the rate of reductive activation of NADP-malate dehydrogenase.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Intrasteric inhibition in redox signalling: light activation of NADP-malate dehydrogenase

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH, EC 1.1.1.82) is inactive in the dark and activated in the light via a reduction of specific disulfides by thiol-disulfide interchange with thioredoxin, reduced by the photosynthetic electron transfer. Compared to the constitutively active NAD-dependent forms, NADP-MDH exhibits two regulatory disulfides per subunit, one located in an N-terminal extension and the other in a C-terminal extension. Convergent information gathered from biochemical, site-directed mutagenesis and structural approaches allowed to solve almost completely the activation mechanism. In the oxidized enzyme, the C-terminal extension is pulled back by the disulfide bridge toward the active-site cleft where the penultimate C-terminal glutamate interacts with one of the arginines involved in substrate binding, thus acting as an internal inhibitor obstructing the access of oxaloacetate. The N-terminal extensions are located at the subunit interface area and rigidify the overall structure of the dimer. Their reduction by reduced thioredoxin triggers a conformational change of the active site towards high-activity conformation, whereas the reduction of the C-terminal bridge expells the C-terminal end from the active site, thus opening the way for the substrate. This revised version was published online in June 2006 with corrections to the Cover Date.     

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.     

The role of active site arginines of sorghum NADP-malate dehydrogenase in thioredoxin-dependent activation and activity

The activation of sorghum NADP-malate dehydrogenase is initiated by thiol/disulfide interchanges with reduced thioredoxin followed by the release of the C-terminal autoinhibitory extension and a structural modification shaping the active site into a high efficiency and high affinity for oxaloacetate conformation. In the present study, the role of the active site arginines in the activation and catalysis was investigated by site-directed mutagenesis and arginyl-specific chemical derivatization using butanedione. Sequence and mass spectrometry analysis were used to identify the chemically modified groups. Taken together, our data reveal the involvement of Arg-134 and Arg-204 in oxaloacetate coordination, suggest an indirect role for Arg-140 in substrate binding and catalysis, and clearly confirm that Arg-87 is implicated in cofactor binding. In contrast with NAD-malate dehydrogenase, no lactate dehydrogenase activity could be promoted by the R134Q mutation. The decreased susceptibility of the activation of the R204K mutant to NADP and its increased sensitivity to the histidine-specific reagent diethylpyrocarbonate indicated that Arg-204 is involved in the locking of the active site. These results are discussed in relation with the recently published NADP-MDH three-dimensional structures and the previously established three-dimensional structures of NAD-malate dehydrogenase and lactate dehydrogenase.     

Direct NMR observation of the thioredoxin-mediated reduction of the chloroplast NADP-malate dehydrogenase provides a structural basis for the relief of autoinhibition

The chloroplastic NADP-dependent malate dehydrogenase (NADP-MDH) catalyzing the reduction of oxaloacetate into L-malate is regulated by light. Its activation results from the thioredoxin-mediated reduction of two disulfides, located, respectively, in N- and C-terminal sequence extensions typical of all NADP-dependent light-regulated forms. Site-directed mutagenesis studies and the resolution of the three-dimensional structure of the oxidized (inactive) Sorghum vulgare enzyme showed that the C-terminal Cys(365)-Cys(377) disulfide constrains the C-terminal extension to fold into the active site where it acts as an internal inhibitor. In the present study, two-dimensional proton NMR spectra of an engineered NADP-MDH rendered monomeric by a 33-amino acid deletion at the N terminus (38 kDa) revealed that a 15-amino acid-long C-terminal peptide (Ala(375) to C-terminal Val(389)) acquired an increased mobility upon reduction, allowing its direct sequence-specific NMR assignment. The location of the flexible peptide in the sequence suggests that the first part of the C-terminal peptide is still folded near the core of the enzyme, so that cysteines 365 and 377 remain in proximity to allow for an efficient reoxidation/inactivation of the enzyme.     

Structural basis for leucine-induced allosteric activation of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) catalyzes reversible conversion between glutamate and 2-oxoglutarate using NAD(P)(H) as a coenzyme. Although mammalian GDH is regulated by GTP through the antenna domain, little is known about the mechanism of allosteric activation by leucine. An extremely thermophilic bacterium, Thermus thermophilus, possesses GDH with a unique subunit configuration composed of two different subunits, GdhA (regulatory subunit) and GdhB (catalytic subunit). T. thermophilus GDH is unique in that the enzyme is subject to allosteric activation by leucine. To elucidate the structural basis for leucine-induced allosteric activation of GDH, we determined the crystal structures of the GdhB-Glu and GdhA-GdhB-Leu complexes at 2.1 and 2.6 Å resolution, respectively. The GdhB-Glu complex is a hexamer that binds 12 glutamate molecules: six molecules are bound at the substrate-binding sites, and the remaining six are bound at subunit interfaces, each composed of three subunits. The GdhA-GdhB-Leu complex is crystallized as a heterohexamer composed of four GdhA subunits and two GdhB subunits. In this complex, six leucine molecules are bound at subunit interfaces identified as glutamate-binding sites in the GdhB-Glu complex. Consistent with the structure, replacement of the amino acid residues of T. thermophilus GDH responsible for leucine binding made T. thermophilus GDH insensitive to leucine. Equivalent amino acid replacement caused a similar loss of sensitivity to leucine in human GDH2, suggesting that human GDH2 also uses the same allosteric site for regulation by leucine.     

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP⁺ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653–658) the total sequence is now known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713–722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme ss characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.     

Effect of high light intensities on oxygen evolution and the light activation of NADP-malate dehydrogenase in intact spinach chloroplasts

The factors limiting the photosynthetic carbon metabolism of intact spinach (Spinacia oleracea L.) chloroplasts after a high-light pretreatment have been studied. Photosynthetic CO₂ fixation was decreased and became more sensitive to the inhibitory effect of the cyclic-electron-flow inhibitor, antimycin A. Depending on the extent of photoinhibition, changing the balance of linear to cyclic electron flow by adding oxaloacetate and antimycin A either did not relieve, or partially relieved the photoinhibitory effect. The decrease in CO₂ fixation appeared to be the consequence of either a limitation by photosystem-II activity (in the case of moderate inhibition) or, at least partially an unfavourable balance between the linear and cyclic electron flows (in the case of strong inhibition). The light activation of NADP-malate dehydrogenase (EC 1.1.1.82) was decreased only in the presence of CO₂, i.e. when there was strong competition for reducing power; otherwise, it was unaffected by photoinhibitory treatments, in accordance with its low energy requirement.Abbreviations Chl chlorophyll - NADP-MDH NADP-dependent malate dehydrogenase - PFD photon flux density - PSI, II photosystem I, II     

Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase.

The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.     

The dark (oxidized) form of the light-activatable NADP-malate dehydrogenase from pea chloroplasts is catalytically active in the presence of guanidine-HCl

The dark form of NADP-malate dehydrogenase from pea leaves which has been shown to contain one disulfide bridge per subunit does not exhibit any catalytic activity in the absence of thiol reducing agents. Upon reduction of these disulfide bridges the enzyme becomes catalytically active. In this presentation, however, it is shown that the oxidized dark form of NADP-malate dehydrogenase becames catalytically competent when assayed in the presence of 200–250 mM guanidine-HCl. This guanidine-dependent activity of the oxidized enzyme is characterized by higher apparent K_m values for the substratres as compared to the reduced enzyme, but is still specific for NADPH. Up to 25% of the V_maxOf the reduced enzyme was obtained for the oxidized guanidine-activated NADP-malate dehydrogenase. The results suggest that the reduction of the regulatory disulfide is not essential for catalytic activity.     

Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity

The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.     

Primary structure of the light-dependent regulatory site of corn NADP-malate dehydrogenase

The light-activated NADP-malate dehydrogenase (NADP-MDH) catalyzes the reduction of oxaloacetate to malate in higher plant chloroplasts. This enzyme is regulated in vivo by the ferredoxin-thioredoxin system through redox reactions. NADP-MDH has been photoactivated in vitro in a chloroplast system reconstituted from the pure protein components and thylakoid membranes. Photoactivation was accompanied by the appearance of new thiol groups (followed by [14C]iodoacetate incorporation). 14C-Carboxymethylated NADP-MDH has been purified from the incubation mixture and its amino-terminal sequence analyzed. Two [14C]carboxymethylcysteines were identified at positions 10 and 15 after light activation, while they were not detected in the dark-treated protein. In addition, the analysis of the tryptic digest of light-activated [14C]carboxymethylated NADP-MDH revealed that the radioactive label was mostly incorporated in Cys10 and Cys15, indicating that these 2 residues play a major role in the light activation mechanism. Moreover, an activation model, in which photoreduced thio-redoxin was replaced by the dithiol reductant dithio-threitol, has been developed. When NADP-MDH was activated in this way, the same sulfhydryls were found to be labeled, and alternatively, they did not incorporate any radioactivity when dithiothreitol reduction was performed after carboxymethylation in denaturating conditions. These results indicate that activation (by light or by dithiothreitol) proceeds on each subunit by reduction of a disulfide bridge located at the amino terminus of the enzyme between Cys10 and Cys15.     

Chloroplast NADP-malate dehydrogenase: structural basis of light-dependent regulation of activity by thiol oxidation and reduction

BACKGROUND: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C4 pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. RESULTS: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 A resolution. The core structure is homologous to AND-dependent malate dehydrogenases. Two surface-exposed and thioredoxin-accessible disulfide bonds are present, one in the N-terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP+, hydrogen bonding to the catalytic His225 as well as obstructing access of the C4 acid substrate. Two loops flanking the active site, termed the Arg2 and Trp loops, that contain the C4 acid substrate binding residues are prevented from closing by the C-terminal extension. CONCLUSIONS: The structure explains the role of the C-terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP+ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous alpha-hydroxy acid dehydrogenases. NADP+ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP+ but not NADPH.     

The dimer contact area of sorghum NADP-malate dehydrogenase: role of aspartate 101 in dimer stability and catalytic activity

During thioredoxin-mediated activation of chloroplastic NADP-malate dehydrogenase, a homodimeric enzyme, the interaction between subunits is known to be loosened but maintained. A modeling of the 3D structure of the protein identified Asp-101 as being potentially involved in the association between subunits through an electrostatic interaction. Indeed, upon site-directed substitution of Asp-101 by an asparagine, the mutated enzyme behaved mainly as a monomer. The mutation strongly affected the catalytical efficiency of the enzyme. The now available 3D structure of the enzyme shows that Asp-101 is protruding at the dimer interface, interacting with Arg-268 of the neighbouring subunit.     

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

Structural basis for catalytic activation of a serine recombinase

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 ? crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggesting roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.     

Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant

To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant.     

Enzyme regulation in C4 photosynthesis: Mechanism of activation of NADP-malate dehydrogenase by reduced thioredoxin

The mechanism of activation of thioredoxin-linked NADP-malate dehydrogenase was investigated by using ¹⁴C-iodoacetate and ¹⁴C-dansylated thioredoxin m, and Sepharose affinity columns (thioredoxin m, NADP-malate dehydrogenase) as probes to monitor enzyme sulfhydryl status and enzyme-thioredoxin interaction. The data indicate that NADP-malate dehydrogenase, purified to homogeneity from corn leaves, is activated by a net transfer of reducing equivalents from thioredoxin m, reduced by dithiothreitol, to enzyme disulfide groups, thereby yielding oxidized thioredoxin m and reduced enzyme. The appearance of new sulfhydryl groups that accompanies the activation of NADP-malate dehydrogenase appears to involve a structural change that is independent of the formation of a stable complex between the enzyme and reduced thioredoxin m. The data are consistent with the conclusion that oxygen promotes deactivation of NADP-malate dehydrogenase through oxidation of SH groups on reduced thioredoxin and on the reduced (activated) enzyme.