The streptococcal flavoprotein NADH oxidase. II. Interactions of pyridine nucleotides with reduced and oxidized enzyme forms

Anaerobic addition of 0.5 eq of NADH/FAD to the streptococcal NADH oxidase produces a redox form spectrally similar to that obtained with 0.5 eq of dithionite/FAD. The second phase of the titration, however, in addition to reducing the flavin with 1 eq of NADH/FAD, leads to the appearance of a long-wavelength absorbance band centered at 725 nm. Reductive titrations of the enzyme with 3-acetylpyridine-adenine dinucleotide, which has a redox potential 72 mV more positive than that of NADH, yield a similar reduced enzyme species. Dithionite reduction of the NADH oxidase followed by titration with NAD+ partially mimics the long-wavelength absorbance of the NADH-reduced enzyme but also leads to the oxidation of 1 FADH2/dimer. NADH is not formed, however, and a similar result is obtained when the dithionite-reduced oxidase is titrated with the nonreducible substrate analog 3-aminopyridine-adenine dinucleotide. These data indicate that the FADH2 oxidation observed is intramolecular and suggest that the active centers of the two apparently identical subunits/dimer are not equivalent. These results also demonstrate that bound pyridine nucleotides can modulate the redox manifold of the NADH oxidase and, when taken together with the effects of these ligands on pre-steady-state behavior, suggest an important regulatory aspect of the catalytic redox function of this unique flavoprotein.     

Equilibrium analyses of the active-site asymmetry in enterococcal NADH oxidase: role of the cysteine-sulfenic acid redox center

Recent studies [Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790-8802] of the O2 reactivity of C42S NADH oxidase (O2 --> H2O2) revealed an asymmetric mechanism in which the two FADH2.NAD+ per reduced dimer display kinetic inequivalence. In this report we provide evidence indicating that the fully active, recombinant wild-type oxidase (O2 --> 2H2O) displays thermodynamic inequivalence between the two active sites per dimer. Using NADPH to generate the free reduced wild-type enzyme (EH2'/EH4), we have shown that NAD+ titrations lead to differential behavior as only one FADH2 per dimer binds NAD+ tightly to give the charge-transfer complex. The second FADH2, in contrast, transfers its electrons to the single Cys42-sulfenic acid (Cys42-SOH) redox center, which remains oxidized during the reductive titration. Titrations of the reduced NADH oxidase with oxidized 3-acetylpyridine and 3-aminopyridine adenine dinucleotides further support the conclusion that the two FADH2 per dimer in wild-type enzyme can be described as distinct "charge-transfer" and "electron-transfer" sites, with the latter site giving rise to either intramolecular (Cys42-SOH) or bimolecular (pyridine nucleotide) reduction. The reduced C42S mutant is not capable of intramolecular electron transfer on binding pyridine nucleotides, thus confirming that the Cys42-SOH center is in fact the source of the redox asymmetry observed with wild-type oxidase. These observations on the role of Cys42-SOH in the expression of thermodynamic inequivalence as observed in wild-type NADH oxidase complement the previously described kinetic inequivalence of the C42S mutant; taken together, these results provide the overlapping framework for an alternating sites cooperativity model of oxidase action.     

Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.     

The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis

Lipoamide dehydrogenase catalyses the NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains a tightly, but noncovalently, bound FAD and a redox-active disulfide, which cycle between the oxidized and reduced forms during catalysis. The mechanism of reduction of the Mycobacterium tuberculosis lipoamide dehydrogenase by NADH and [4S-(2)H]-NADH was studied anaerobically at 4 degrees C and pH 7.5 by stopped-flow spectrophotometry. Three phases of enzyme reduction were observed. The first phase, characterized by a decrease in absorbance at 400-500 nm and an increase in absorbance at 550-700 nm, was fast (k(for) = 1260 s(-)(1), k(rev) = 590 s(-)(1)) and represents the formation of FADH(2).NAD(+), an intermediate that has never been observed before in any wild-type lipoamide dehydrogenase. A primary deuterium kinetic isotope effect [(D)(k(for) + k(rev)) approximately 4.2] was observed on this phase. The second phase, characterized by regain of the absorbance at 400-500 nm, loss of the 550-700 nm absorbance, and gain of 500-550 nm absorbance, was slower (k(obs) = 200 s(-)(1)). This phase represents the intramolecular transfer of electrons from FADH(2) to the redox-active disulfide to generate the anaerobically stable two-electron reduced enzyme, EH(2). The third phase, characterized by a decrease in absorbance at 400-550 nm, represents the formation of the four-electron reduced form of the enzyme, EH(4). The observed rate constant for this phase showed a decreasing NADH concentration dependence, and results from the slow (k(for) = 57 s(-)(1), k(rev) = 128 s(-)(1)) isomerization of EH(2) or slow release of NAD(+) before rapid NADH binding and reaction to form EH(4). The mechanism of oxidation of EH(2) by NAD(+) was also investigated under the same conditions. The 530 nm charge-transfer absorbance of EH(2) shifted to 600 nm upon NAD(+) binding in the dead time of mixing of the stopped-flow instrument and represents formation of the EH(2).NAD(+) complex. This was followed by two phases. The first phase (k(obs) = 750 s(-)(1)), characterized by a small decrease in absorbance at 435 and 458 nm, probably represents limited accumulation of FADH(2).NAD(+). The second phase was characterized by an increase in absorbance at 435 and 458 nm and a decrease in absorbance at 530 and 670 nm. The observed rate constant that describes this phase of approximately 115 s(-)(1) probably represents the overall rate of formation of E(ox) and NADH from EH(2) and NAD(+), and is largely determined by the slower rates of the coupled sequence of reactions preceding flavin oxidation.     

The non-flavin redox center of the streptococcal NADH peroxidase. I. Thiol reactivity and redox behavior in the presence of urea

Unlike the 2-electron-reduced (EH2) forms of the flavoprotein disulfide reductases and mercuric reductase, the native EH2 form of the streptococcal NADH peroxidase is quite refractile toward chemical modification with thiol-specific reagents. In the presence of 1.3 M urea, however, the single thiol of the reduced enzyme reacts with phenylmercuric acetate with a t1/2 of 3 min. This modification abolishes the charge-transfer absorbance band at 540 nm and inactivates the enzyme; the latter effect is shown to be reversed with dithiothreitol. Alkylation of the streptococcal peroxidase with iodo[1-14C]acetamide under reducing conditions in the presence of 8 M guanidine hydrochloride allows the isolation of a single labeled tryptic peptide with the sequence: Gly-Asp-Phe-Ile-Ser-Phe-Leu-Ser-C*ys-Gly-Met-Gln-Leu-Tyr-Leu- Glu-Gly-Lys. This sequence is identical to that previously reported (Poole, L. B., and Claiborne, A. (1988) Biochem. Biophys. Res. Commun. 153, 261-266) for the cysteinyl peptide isolated from the NADH peroxidase labeled metabolically with [35S]cysteine. Careful examination of the physical properties of the streptococcal peroxidase in the presence of 1.3 M urea shows that, while catalytic activity and native structural features are largely retained, the relative potentials of flavin and non-flavin redox centers are dramatically affected. We propose that low concentrations of urea stabilize an intermediate state in the transition between native and denatured forms, which is responsible for the observed changes in both active-site thiol reactivity and in redox properties.     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

Switching pyridine nucleotide specificity in P450 BM3: mechanistic analysis of the W1046H and W1046A enzymes

Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K(m)(NADPH)/k(cat)/K(m)(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant approximately 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

Catalytic properties of streptococcal NADH oxidase containing artificial flavins.

The flavoprotein NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the tetravalent reduction of O2-->2H2O, has been purified as the apoenzyme to allow reconstitution studies with both native and artificial flavins. Turnover numbers for the enzyme containing 1-deaza-, 2-thio-, and 4-thio-FAD range from 51 to 4% of that of the native FAD enzyme; these reconstituted oxidases also catalyze the four-electron reduction of oxygen. Dithionite and NADH titrations of the native FAD oxidase require 1.7 eq of reductant/FAD and follow spectral courses very similar to those previously reported for the purified holoenzyme. Azide is a linear mixed-type inhibitor with respect to NADH, and dithionite titrations in the presence of azide yield significant stabilization of the neutral blue semiquinone. Redox stoichiometries for the oxidase containing modified flavins range from 1.1 to 1.4 eq of reductant/FAD. Spectrally distinct reduced enzyme.NAD+ complexes result with all but the 2-thio-FAD enzyme on titration with NADH. The reduced 4-thio-FAD oxidase shows little or no evidence of desulfurization to native FAD on reduction and reoxidation. Both the 8-mercapto- (E'o = -290 mV) and 8-hydroxy-FAD (E'o = -335 mV) oxidase are readily reduced by excess NADH. These results offer a further basis for analysis of the active-site structure and oxygen reactivity of this unique flavoprotein oxidase.     

The streptococcal flavoprotein NADH oxidase. I. Evidence linking NADH oxidase and NADH peroxidase cysteinyl redox centers

The FAD-containing NADH oxidase from Streptococcus faecalis 10C1, which catalyzes the four-electron reduction of O2----2H2O, has been purified by an improved procedure for analyses of its structural and redox properties. The enzyme is apparently a dimer of two identical subunits, each containing 1 mol of FAD. Dithionite reduction of the enzyme proceeds in two distinct phases corresponding to approximately 0.5 and 1.1 eq/FAD, respectively. Thiol assays of the NADH oxidase, reduced anaerobically with 1 eq of NADH/FAD prior to denaturation, are consistent with the presence of a single redox-active cysteinyl residue/subunit. Analysis of the cysteinyl peptides of the oxidase, identified in tryptic digests of the enzyme labeled metabolically with [35S]cysteine, reveals a sequence which is closely related to the redox-active cysteinyl peptide sequence recently determined for the streptococcal flavoprotein NADH peroxidase. A second cysteinyl peptide sequence, when aligned with residues 3-17 of the peroxidase NH2-terminal sequence, reveals identity in 7 of 15 positions and satisfies several of the criteria described for ADP-binding structures. Additional probes of the structural and redox properties of the NADH oxidase, including visible circular dichroism spectroscopy and sensitivity to inactivation by hydrogen peroxide, provide further evidence for a fundamental structural connection between flavin-dependent NADH oxidase and peroxidase functions.     

Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure

The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.     

Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart

The effect of NAD+ on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH2) couple in the presence on NAD+ were -218 mV and -251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD+ binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD+. Fluorescence quenching of bound FAD by NAD+ was released by phenobarbital. The results suggest that NAD+ strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD+ acts not only as an acceptor of electrons from EH2, but also as an effector in the flavin-disulfide interaction of EH2.     

Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases

Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.     

Purification and properties of the glutathione reductase of Chromatium vinosum.

The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.     

Interaction of glutathione reductase with heavy metal: the binding of Hg(II) or Cd(II) to the reduced enzyme affects both the redox dithiol pair and the flavin

To determine the inhibition mechanism of yeast glutathione reductase (GR) by heavy metal, we have compared the electronic absorption and resonance Raman (RR) spectra of the enzyme in its oxidized (Eox) and two-electron reduced (EH2) forms, in the absence and the presence of Hg(II) or Cd(II). The spectral data clearly show a redox dependence of the metal binding. The metal ions do not affect the absorption and RR spectra of Eox. On the contrary, the EH2 spectra, generated by addition of NADPH, are strongly modified by the presence of heavy metal. The absorption changes of EH2 are metal-dependent. On the one hand, the main flavin band observed at 450 nm for EH2 is red-shifted at 455 nm for the EH2-Hg(II) complex and at 451 nm for the EH2-Cd(II) complex. On the other hand, the characteristic charge-transfer (CT) band at 540 nm is quenched upon metal binding to EH2. In NADPH excess, a new CT band is observed at 610 nm for the EH2-Hg(II)-NADPH complex and at 590 nm for EH2-Cd(II)-NADPH. The RR spectra of the EH2-metal complexes are not sensitive to the NADPH concentration. With reference to the RR spectra of EH2 in which the frequencies of bands II and III were observed at 1582 and 1547 cm-1, respectively, those of the EH2-metal complexes are detected at 1577 and 1542 cm-1, indicating an increased flavin bending upon metal coordination to EH2. From the frequency shifts of band III, a concomitant weakening of the H-bonding state of the N5 atom is also deduced. Taking into account the different chemical properties of Hg(II) and Cd(II), the coordination number of the bound metal ion was deduced to be different in GR. A mechanism of the GR inhibition is proposed. It proceeds primarily by a specific binding of the metal to the redox thiol/thiolate pair and the catalytic histidine of EH2. The bound metal ion then acts on the bending of the isoalloxazine ring of FAD as well as on the hydrophobicity of its microenvironment.     

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.     

Carboxymethylated liver alcohol dehydrogenase: kinetic and thermodynamic characterization of reactions with substrates and inhibitors

Liver alcohol dehydrogenase (LADH) carboxymethylated at Cys-46 (CMLADH) forms two different ternary complexes with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA). The complex with reduced nicotinamide adenine dinucleotide (NADH) is characterized by a 38-nm red shift of the long-wavelength pi, pi* transition to 436 nm, while the complex with oxidized nicotinamide adenine dinucleotide (NAD+) is characterized by a 60-nm red shift to 458 nm. CMLADH also forms a ternary complex with NAD+ and the Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime in which the absorption of the oxime (lambda max = 354 nm) is red shifted 80 nm to 434 nm. Pyrazole and 4-methylpyrazole are weak competitive inhibitors of ligand binding to the substrate site of native LADH. These inhibitors were found to form ternary complexes with CMLADH and NADH which are more stable than the corresponding complexes with the native enzyme. The transient reductions of the aldehydes DACA and p-nitrobenzaldehyde (NBZA) were studied under single-turnover conditions. Carboxymethylation decreases the DACA reduction rate 80-fold and renders the process essentially independent of pH over the region 5-9, whereas this process depends on a pKa of 6.0 in the native enzyme. At pH 7.0, the rate constant for NBZA reduction also is decreased at least 80-fold to a value of 7.7 +/- 0.3 s-1. Since primary kinetic isotope effects are observed when NADH is substituted with (4R)-4-deuterio-NADH (kH/kD = 3.0 for DACA and kH/kD = 2.3 for NBZA), the rate-limiting step for both aldehydes involves hydride transfer. The altered pH dependence is concluded to be due to an increase in the pK value of the zinc-coordinated DACA-alcohol in the ternary complex with NAD+ by more than 3 units. This perturbation is brought about by the close proximity of the negatively charged carboxymethyl carboxylate.     

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salts- induced oxidase activity of lipoamide dehydrogenase from pig heart.

A weak NADH oxidase activity of lipoamide dehydrogenase at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3 to 5.5 without changing the maximum activity. The optimum pH is similarly shifted to 5.6 when sulfhyldryl groups of the enzyme are oxidized in the presence of small amount of cupric ion. The NADH: lipoamide and NADH: p-benzoquinone reductase activities are strongly inhibited by KI but both are increased by the presence of (NH4)2SO4. The known intermediate having a charge-transfer band at 530 nm can be seen upon an addition of NADH to the enzyme in the presence of (NH4)2SO4 but not in the presence of KI. The enzyme flavin is reductase by a stoichiometric amount of NADH when KI is present.