The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.     

A negative cooperativity between NADPH and adrenodoxin on binding to NADPH:adrenodoxin reductase

Adrenodoxin stimulated the oxidation of NADPH by 1,4-benzoquinone, catalyzed by NADPH:adrenodoxin reductase. It prevented the enzyme inhibition by NADPH and formed an additional pathway of benzoquinone reduction presumably via reduced adrenodoxin. In the presence of 100-400 microM NADP+, which increased the Km of NADPH, adrenodoxin acted as a partial competitive inhibitor for NADPH decreasing its TN/Km by a limiting factor of 3. Ki of adrenodoxin decreased on the NADP+ concentration decrease and was estimated to be about 10(-8) M in the absence of NADP+.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Nitrate reductase from Penicillium chrysogenum. Purification and kinetic mechanism

Nitrate reductase (NADPH:nitrate oxidoreductase; EC 1.6.6.1-3) was purified to apparent homogeneity from mycelium of Penicillium chrysogenum. The final preparation catalyzed the NADPH-dependent, FAD-mediated reduction of nitrate with a specific activity of 170-225 units X mg of protein-1. Gel filtration and glycerol density centrifugation yielded, respectively, a Stokes radius of 6.3 nm and an s20,w of 7.4. The molecular weight was calculated to be 199,000. On sodium dodecyl sulfate gels, the enzyme displayed two almost contiguous dye-staining bands corresponding to molecular weights of about 97,000 and 98,000. The enzyme prefers NADPH to NADH (kspec ratio = 2813), FAD to FMN (kspec ratio = 141), FAD (+ NADPH) to FADH2 (kspec ratio = 12,000), and nitrate to chlorate (kspec ratio = 4.33), where the kspec (the specificity constant for a given substrate) represents Vmax/Km. The Penicillium enzyme will also catalyze te NADPH-dependent, FAD-mediated reduction of cytochrome c with a specific activity of 647 units X mg of protein-1 (Kmcyt = 1.25 X 10(-5) M), and the reduced methyl viologen (MVH2, i.e. methyl viologen + dithionite)-dependent, NADPH and FAD-independent reduction of nitrate with a specific activity of 250 units X mg of protein-1 kmMVH2 = 3.5 X 10(-6) M). Initial velocity studies showed intersecting NADPH-FAD and nitrate-FAD reciprocal plot patterns. The NADPH-nitrate pattern was a series of parallel lines at saturating and unsaturating FAD levels. NADP+ was competitive with NADPH, uncompetitive with nitrate (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. Nitrite was competitive with nitrate, uncompetitive with NADPH (at saturating and unsaturating FAD levels), and a mixed-type inhibitor with respect to FAD. At unsaturating nitrate and FAD, NADPH exhibited substrate inhibition, perhaps as a result of binding to the FAD site(s). At very low FAD concentrations, low concentrations of NADP+ activated the reaction slightly. The initial velocity and product inhibition patterns are consistent with either of the two kinetic mechanisms. One (rather unlikely) mechanism involves the rapid equilibrium random binding of all ligands with (a) NADP+ and NADPH mutually exclusive, (b) nitrate and nitrite mutually exclusive, (c) the binding of NADPH strongly inhibiting the binding of nitrate and vice versa, (d) the binding of NADPH strongly promoting the binding of nitrite and vice versa, and (e) the binding of nitrate strongly promoting the binding of NADP+ and vice versa...     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k(ca)(t)/K(m) value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH(4)) and two-electron reduced (EH(2)) states in quinone reduction. The redox potential of the EH(2)/EH(4) couple of TrxR calculated according to the Haldane relationship with NADPH/NADP(+) was -0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K(i) = 6.3 +/- 1 microm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.     

Steady-state and laser flash induced photoreduction of yeast glutathione reductase by 5-deazariboflavin and by a viologen analogue: stabilization of flavin adenine dinucleotide semiquinone species by complexation

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).     

The rotenone-insensitive reduction of quinones and nitrocompounds by mitochondrial NADH:ubiquinone reductase

The rotenone-insensitive reduction of quinones and aromatic nitrocompounds by mitochondrial NADH: ubiquinone reductase (complex I, EC 1.6.99.3) has been studied. It was found that these reactions proceed via a mixed one- and two-electron transfer. The logarithms of the bimolecular rate constants of oxidation (TN/Km) are proportional to the one-electron-reduction potentials of oxidizers. The reactivities of nitrocompounds are close to those of quinones. Unlike the reduction of ferricyanide, these reactions are not inhibited by NADH. However, they are inhibited by NAD+ and ADP-ribose, which also act as the mixed-type inhibitors for ferricyanide. TN/Km of quinones and nitrocompounds depend on the NAD+/NADH ratio, but not on NAD+ concentration. They are diminished by the limiting factors of 2.5-3.5 at NAD+/NADH greater than 200. It seems that rotenone-insensitive reduction of quinones and nitrocompounds takes place near the NAD+/NADH and ferricyanide binding site, and the inhibition is caused by induced conformational changes after the binding of NAD+ or ADP-ribose.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.     

A 'branched' mechanism of the reverse reaction of yeast glutathione reductase. An estimation of the enzyme standard potential values from the steady-state kinetics data

The reduced glutathione-linked NADP+ reduction, catalyzed by yeast glutathione reductase, follows a 'sequential' or 'ping-pong' mechanism at high or low NADP+ concentrations, respectively. The pattern of the NADPH and NADP+ cross-inhibition reflects not only the competition for the binding site, but the shift of the reaction equilibrium as well. A 'branched' scheme of the glutathione reductase reaction is presented. The enzyme standard potential (-255 mV, pH 7.0) was estimated from the ratio of the NADPH and NADP+ rate constants corresponding to the ping-pong mechanism.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.     

Activation of mercuric reductase by the substrate NADPH

Preincubation of the oxidized form of the flavoenzyme mercuric reductase with the reducing substrate, NADPH, or with a high concentration of cysteine (30 mM) results in a substantial increase of the catalytic activity as measured in a standard spectrophotometric assay. Also NADH has some activating effect but NADP+ or EDTA have no effect. In the presence of 1 mM cysteine only one equivalent of NADPH per FAD seems to be required for full activation which occurs after an incubation time of about 10 min. Activated mercuric reductase appears to be stable under anaerobic conditions but eventually returns to the original level of activity in the presence of oxygen. The activated state seems to be stabilized by 1 mM cysteine. Activation of mercuric reductase does not seem to be correlated with a change in the number of reactive thiol groups. The chemical nature of the activation process is not yet understood. Stopped-flow studies have shown that the nonactivated enzyme is practically inactive prior to contact with the substrates. The enzyme is gradually activated during the assay. The kinetics of activation of the 'native' enzyme is biphasic but 'clipped' enzyme, lacking an 85-residue N-terminal domain, is activated in a single first-order process. The progress curves obtained with preactivated enzyme are approximately exponential even at saturating concentrations of NADPH (Km = 0.4 microM at 25 degrees C, pH 7.3) and Hg2+ (Km = 3.2 microM in the presence of 1 mM cysteine). The initial rates yield kcat values of about 13 s-1 per FAD molecule (25 degrees C, pH 7.3). We find no evidence for a thiol-dependent change from a rapid to a slow kinetic phase. The shape of the progress curves presumably depends on product inhibition, but NADP+ is not a sufficiently effective inhibitor to explain the effect fully.     

Electronic energy transfer and fluorescence quenching in the active sites of mercuric reductase

The FAD-containing enzyme mercuric reductase has been studied by means of steady-state and time-resolved fluorescence spectroscopy. The fluorescence relaxation of the excited state of the isoalloxazine ring of FAD can be described by a sum of two exponential functions. The two lifetimes are not due to a different lifetime of each of the two FAD molecules of mercuric reductase. The FAD molecules are quenched dynamically by a quencher that is not sensitive to the solvent viscosity. In vitro activation induces a dynamic quenching of fluorescence, while upon binding of NADP+ the FAD molecules are both statically and dynamically quenched. Time-resolved fluorescence anisotropy experiments of mercuric reductase in water show that the isoalloxazine ring probably undergoes a rapid and restricted vibrational motion of small amplitude. Electronic energy transfer occurs between the two FAD molecules at a rate of about 3.4 x 10(7) s-1. The angle between the emission transition dipole of the donor and the absorption transition dipole of the acceptor is 137 +/- 2 degrees (or 43 +/- 2 degrees). From previous X-ray data of glutathione reductase we find that the corresponding angle is 160 degrees. This suggests that the isoalloxazine rings of mercuric reductase and glutathione reductase are mutually tilted in slightly different ways.     

Determination of the redox properties of human NADPH-cytochrome P450 reductase

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.     

Purification and properties of heterodisulfide reductase from Methanobacterium thermoautotrophicum (strain Marburg)

The reduction of the heterodisulfide of coenzyme M (H-S-CoM) and 7-mercaptoheptanoyl-L-threonine phosphate (H-S-HTP) is a key reaction in the metabolism of methanogenic bacteria. The heterodisulfide reductase catalyzing this step was purified 80-fold to apparent homogeneity from Methanobacterium thermoautotrophicum. The native enzyme showed an apparent molecular mass of 550 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of three different subunits of apparent molecular masses 80 kDa, 36 kDa, and 21 kDa. The enzyme, which was brownish yellow, contained per mg protein 7 +/- 1 nmol FAD, 130 +/- 10 nmol non-heme iron and 130 +/- 10 nmol acid-labile sulfur, corresponding to 4 mol FAD and 72 mol FeS/mol native enzyme. The purified heterodisulfide reductase catalyzed the reduction of CoM-S-S-HTP (app. Km = 0.1 mM) with reduced benzylviologen at a specific rate of 30 mumol.min-1.mg protein-1 (kcat = 68 s-1) and the reduction of methylene blue with H-S-CoM (app. Km = 0.2 mM) plus H-S-HTP (app. Km less than 0.05 mM) at a specific rate of 15 mumol.min-1.mg-1. The enzyme was highly specific for CoM-S-S-HTP and H-S-CoM plus H-S-HTP. The physiological electron donor/acceptor remains to be identified.     

FAD analogues as prosthetic groups of human glutathione reductase. Properties of the modified enzyme species and comparisons with the active site structure

Human glutathione reductase (NADPH + GSSG + H+ in equilibrium with NADP+ + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD greater than 8-azido-FAD = 8-F-FAD = 8-C1-FAD greater than 4-thio-FAD = 1-deaza-FAD greater than 2-thio-FAD. The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD X glutathione reductase was crystallized for X-ray diffraction analysis. Detailed studies were focussed on position 8 of the flavin. 8-Cl-FAD X glutathione reductase and 8-F-FAD X glutathione reductase reacted only poorly with HS- to give 8-mercapto-FAD X glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD X glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate. 8-Mercapto-FAD X glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the thiolate of Cys63 in the native enzyme is not per se essential for catalysis. The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of alpha-helix 338-354 which starts out close to the N(1)/C(2)/O(2 alpha) region of the flavin. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acryloyl-CoA reductase from Clostridium propionicum. An enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein.

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(alpha2betagamma)4; molecular mass 600 +/- 50 kDa] composed of a propionyl-CoA dehydrogenase (alpha2, 2 x 40 kDa) and an electron-transferring flavoprotein (ETF; beta, 38 kDa; gamma, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per alpha2betagamma subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 +/- 1 microm; kcat = 4.5 s-1 at 100 microm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 microm; kcat = 29 s-1 at 300 microm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 microm; kcat = 2.0 s-1) or butyryl-CoA (Km = 100 microm; kcat = 3.5 s-1) as electron donor and 200 microm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the beta and gamma subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.     

Nonphysiological redox-agents are reduced at the binding center of NADP(H) glutathione reductase]

Studies of the acceptor reductase reaction of yeast glutathione reductase (EC 1.6.4.2) revealed that the competitive inhibitors for NADPH, 2',5'-ADP and Br- decrease the rate constants for the enzyme oxidation by ferricyanide, phenanthrene quinone, and juglone. A similar effect is observed when NADH which does not bind to the reduced enzyme is used as substrate. These observations support the hypothesis that non-physiological redox agents are reduced at the NADP(H)-binding center of glutathione reductase and that NADP(H) binding stimulates the reaction by displacing tyrosine-197 which protects FAD from the solvent.