Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.     

On the catalytic role of the active site residue E121 of E. colil-aspartate oxidase

l-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD⁺. The enzyme oxidizes l-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor.     

Structural and mechanistic mapping of a unique fumarate reductase

The 1.8 A resolution crystal structure of the tetraheme flavocytochrome c3, Fcc3, provides the first mechanistic insight into respiratory fumarate reductases or succinate dehydrogenases. The multi-redox center, three-domain protein shows a 40 A long 'molecular wire' allowing rapid conduction of electrons through a new type of cytochrome domain onto the active site flavin, driving the reduction of fumarate to succinate. In this structure a malate-like molecule is trapped in the enzyme active site. The interactions between this molecule and the enzyme suggest a clear mechanism for fumarate reduction in which the substrate is polarized and twisted, facilitating hydride transfer from the reduced flavin and subsequent proton transfer. The enzyme active site in the oxidized form is completely buried at the interface between the flavin-binding and the clamp domains. Movement of the cytochrome and clamp domains is postulated to allow release of the product.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.     

Oxidation of protoporphyrinogen in the obligate anaerobe Desulfovibrio gigas.

The anaerobic oxidation of protoporphyrinogen to protoporphyrin was demonstrated in extracts of Desulfovibrio gigas. Protoporphyrin formation occurred in the presence of nitrite, hydroxylamine, sulfite, thiosulfate, ATP plus sulfate, NAD+, NADP+, flavin adenine dinucleotide, flavin mononucleotide, fumarate, 2,6-dichlorophenol-indophenol, methyl viologen, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. With dialyzed cell extracts, highest activities were observed with sulfite, NAD+, and NADP+ as electron acceptors. The enzyme for protoporphyrinogen oxidation was localized in the membrane of D. gigas and displayed optimal activity at pH 7.3 and 28 degrees C.     

The NADH dehydrogenase of the respiratory chain of Escherichia coli. II. Kinetics of the purified enzyme and the effects of antibodies elicited against it on membrane-bound and free enzyme.

The purified respiratory chain NADH dehydrogenase of Escherichia coli oxidizes NADH with either dichlorophenolindophenol (DCIP). ferricyanide, or menadione as electron acceptors, with values for NADH are similar with the three electron acceptors (approximately 50 muM). The purified enzyme contains no flavin and has an absolute requirement for FAD, with Km values around 4 muM. The pH optimum of the enzyme appears to be between 6.5 and 7; the optimum is difficult to establish because of nonenzymatic reduction of DCIP at the lower pH values. Potassium cyanide stimulates the DCIP reductase activity about 2-fold, but has no effect on ferricyanide reductase. The enzyme exhibits hyperbolic kinetics with respect to NADH concentration in both the ferricyanide and DCIP reductase assays, but cooperatively is seen in the menadione reductase reaction. NAD+ is an effective competitive inhibitor of the reaction (Ki congruent to 20 muM); in the presence of NAD+, the NADH saturation curve becomes cooperative, even in the DCIP reductase assay. Many adenine containing nucleotides are competitive inhibitors of the enzyme. The apparent Ki values for these nucleotides as inhibitors of the purified enzyme, the membrane-bound NADH dehydrogenase, and the NADH oxidase are equivalent. An examination of inhibitory effects of a series of adenine nucleotides suggests that the inhibitors act as analogues of NAD+, which is the true physiological inhibitor. The results suggest that the enzyme in situ is always partially inhibited by the levels of NAD- in the E coli cell, and thus behaves in a cooperative fashion to changes in the NAD+/NADH ratio. An antibody has been elicited against the purified NADH dehydrogenase. Immunodiffusion and crossed immunoelectrophoresis show that the antibody is directed principally against the NADH dehydrogenase, with some activity against minor contaminants in the purified preparation. The antibody inhibits NADH dehydrogenase activity 50% at saturating levels. When this antibody preparation is used to examine solubilized membrane preparations, two major immunoprecipitates are found. A parallel inhibition of the membrane-bound NADH dehydrogenase and NADH oxidase activities is seen, supporting the hypothesis that the purified enzyme is indeed a component of the respiratory chain-dependent NADH oxidase pathway.     

Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase.

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.     

Acyl-CoA oxidase from Candida tropicalis

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.     

Activation of a flavoprotein by proteolysis

Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax/KM) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme. Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5mM), displayed three bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase. Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.     

Dual role of NADP(H) in the reaction of a flavin dependent N-hydroxylating monooxygenase

Aspergillus fumigatus siderophore A (Af SidA) is a flavin-dependent monooxygenase that catalyzes the hydroxylation of ornithine, producing N(5)-hydroxyornithine. This is the first step in the biosynthesis of hydroxamate-containing siderophores in A. fumigatus. Af SidA is essential for virulence, validating this enzyme as a drug target. Af SidA can accept reducing equivalents from either NADPH or NADH and displays similar kinetic parameters when using either coenzyme. When the enzyme is reduced with NADPH and reacted with molecular oxygen, a C4a-hydroperoxyflavin intermediate is observed. When the enzyme is reduced with NADH, the intermediate is 2-fold less stable. Steady-state kinetic isotope effect values of 3 and 2 were determined for NADPH and NADH, respectively. The difference in the isotope effect values is due to differences in the rate of flavin reduction by these coenzymes. A difference in the binding mode between these coenzymes was observed by monitoring flavin fluorescence. Limited proteolysis studies show that NADP(+), and not NAD(+), protects Af SidA from proteolysis, suggesting that it induces conformational changes upon binding. Together, these results are consistent with NADPH having a role in flavin reduction and in the modulation of conformational changes, which positions NADP(+) to also play a role in stabilization of the C4a-hydroperoxyflavin.     

Effect of coenzymes on the quaternary structure conformation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle.

Kinetics of thermal inactivation of glyceraldehyde-3-phosphate dehydrogenases of mung beans and rabbit muscle have been studied under different pH conditions in the absence and presence of various concentrations of NAD+ and NADH. The data have been discussed with respect to the effect of the coenzymes on the quaternary structure symmetry of the two enzymes and their binding isotherms. Both the (homo-tetrameric) apo-enzymes exhibit biphasic kinetics of thermal inactivation, characteristic of C2 symmetry, at lower pH values and a single exponential decay of enzyme activity, characteristic of D2 symmetry, at higher pHs. In each case, NAD+ has no effect on the biphasic kinetic pattern of thermal inactivation at lower pH values, but NADH brings about a change to single exponential decay. At higher pH values, NADH does not affect the kinetic pattern (single exponential decay) of any enzyme, but NAD+ alters it to biphasic kinetics in each case. The data suggest that NAD+ and NADH have higher affinity for the C2 and D2 symmetry conformation, respectively. With mung beans enzyme, the effect of NAD+ on the two rate constants of biphasic inactivation at pH 7.3 is consistent with a Kdiss equal to 110 microM. The NAD(+)-dependent changes in the kinetic pattern of thermal inactivation of this enzyme at pH 8.6 suggest a positive cooperativity in the coenzyme binding (nH = 3.0). In the binding of NADH to the mung beans enzyme, a weak positive cooperativity is observed at pH 7.3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.     

Rabbit liver pyridoxamine (pyridoxine) 5'-phosphate oxidase. Purification and properties.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been purified 2000-fold from rabbit liver. The enzyme preparation migrates as a single protein and activity band on analytical disc gels containing 4,7, or 9 percent acrylamide, and as a single protein band on sodium dodecyl sulfate acrylamide gels. The oxidase is, therefore, homogeneous by these criteria. The pure enzyme catalyzes the following reactions in the presence of FMN: (See journal for formula). These activities copurify in the ratio of 1:1:1. Apparent K-m values are 10 muM for pyridoxamine-P, 30 muM for pyridoxine-P, and 40 nM for FMN. Apparent K-m values for N-(phosphopyridoxyl)amines range from 3.1 times 10-5 M to 1.6 times 10-3 M. The dissociation constant for FMN binding, determined by quenching of protein fluorescence, is 20 nM. The pH optima for all three types of substrates are broad, with maxima near pH 9. The pH dependence of FMN binding, measured by quenching of flavin fluorescence, has the same shape as the substrate activity profile. The holoenzyme has absorption maxima red-shifted from those of FMN to 380 nm and 448 nm, and exhibits spectral changes typical of flavoproteins upon reduction with dithionite. Its oxidation-reduction potential at pH 7 in phosphate buffer is -0.131 volt. The native enzyme has a molecular weight of 54,000 and is made up of two possibly identical polypeptide chains with molecular weights of 27,000. The applicability of proposed mechanisms of flavin catalysis to this flavoprotein is discussed.     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

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.     

Demonstration and possible function of NADH:NAD+ transhydrogenase from ascaris muscle mitochondria.

Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.     

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.     

One-electron reduction of hepatic NADH-cytochrome b5 reductase as studied by pulse radiolysis

The reduction of flavin in hepatic NADH-cytochrome b5 reductase by the hydrated electron (eaq-) was investigated by pulse radiolysis. The eaq- reduced the flavin of NADH-cytochrome b5 reductase to form the red semiquinone between pH 5 and 9. The spectrum of the red semiquinone differs from that of enzyme reduced by dithionite in the presence of NAD+. After the first phase of the reduction, conversion of the red to blue semiquinone was observed at acidic pH. Resulting products are the blue (neutral) or red (anionic) semiquinone or a mixture of the two forms. The pK value for this flavin radical was approximately 6.3. Subsequently, the semiquinone form reacted by dismutation to form the oxidized and the fully reduced forms of the enzyme with a rate constant of 1 x 10(3) M-1 s-1 at pH 7.1. In the presence of NAD+, eaq- reacted with NAD+ to yield NAD(.). Subsequently, NAD. transferred an electron to NAD+-bound oxidized enzyme to form the blue and red semiquinone or mixture of the two forms of the enzyme, where pK value of this flavin radical was approximately 6.3. The blue semiquinone obtained at acidic pH was found to convert to the red semiquinone with a first order rate constant of 90 s-1, where the rates were not affected by pH or the concentration of NAD+. The final product is NAD+-bound red semiquinone of the enzyme.