The reaction of 8-mercaptoflavins and flavoproteins with sulfite. Evidence for the role of an active site arginine in D-amino acid oxidase

This work presents strong evidence that the role of the active site arginine in D-amino acid oxidase is to act as a positively charged group interacting with the flavin N(1)-C(2) = 0 locus. Modification with cyclohexanedione, which has been shown previously to modify specifically an active site arginine in D-amino acid oxidase (Ferti, C., Curti, B., Simonetta, M. P., Ronchi, S., Galliano, M., and Minchiotti, L. (1981) Eur. J. Biochem. 119, 553-557) destroys the ability of D-amino acid oxidase to stabilize the benzoquinoid type spectrum of 8-mercapto-FAD and destroys the ability to form a flavin N-5 adduct with sulfite. Both of these properties have been attributed to the presence of such a group. The active site lysine, histidine, and tyrosine have been ruled out as possibilities for such a group. In addition, the reactivity of flavoproteins containing 8-mercaptoflavin with sulfite has been examined and falls into the same two general classes as the reactivity of the native flavoproteins: oxidases form N-5 adducts while all of the other 8-mercaptoflavoproteins examined do not, forming instead the 8-sulfonate flavin.     

Properties of flavins where the 8-methyl group is replaced by mercapto- residues.

Sulfur functions in position 8 of the flavin nucleus give rise to new modified flavin derivatives, which should prove useful as probes of the flavin binding domains of flavoproteins. Here, we report on some properties of 8-nor-8-alkylmercaptoflavins and 8-nor-8-mercaptoflavin which are readily formed by nucleophilic displacement by alkylmercaptides or sulfide, with 8-nor-8-chloroflavins as starting material. The new flavins are characterized by extensive shifts in spectral properties, with very high extinction coefficients. 8-nor-8-mercaptoriboflavin is easily and reversibly converted to its (-S-S-) dimer. Oxidation of the sulfur group by peracids forms first sulfoxides and then sulfones, in which the characteristic usual flavin spectrum is regained. A comparison of 8-SR-8-nor-flavins with 8-OR-8-nor-flavins (Ghisla, S., and Mayhew, S.G. (1976) Eur. J. Biochem 63, 373-390) indicates that in both classes of compounds, optical properties, ionization constants, and oxidation-reduction potentials follow similar patterns.     

6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins

6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-SO3--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20 degrees C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiosulfonate, H2O2, and m-chloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate.     

Resonance Raman studies on 8-mercaptoriboflavin

Resonance Raman (RR) spectroscopy on various ionic species of 8-mercaptoriboflavin in aqueous solution shows large vibrational spectral changes upon ionization of the 8-SH group. These changes and the superposition of the RR excitation profile and the visible spectrum for the 8-S^? species indicate that there is a substantial amount of the “quinoid thioketone” resonance form present in aqueous solutions of the 8-S^? mercaptoriboflavin. RR spectra of 8-mercaptoriboflavin bound to riboflavin binding protein confirm the conclusion (reached on the basis of visible spectra) that the 8-mercaptoriboflavin binds to the protein in the 8-SH, protonated, form. There are, however, changes in the 1250-cm^?1 region of the RR spectrum upon binding aqueous 8-mercaptoflavin to the protein. The 1257-cm^?1 band in aqueous solution moves to 1248 cm^?1 on the protein surface. This shift is also observed in DMSO solution of 8-mercaptoriboflavin. Since the 1257-cm^?1 band shifts upon formation of the N-3 deuteroriboflavin, we interpret this lowering of a δ-H mode at N-3 to mean that there is weaker hydrogen bonding between flavin N-3 and riboflavin binding protein than between flavin and water.     

Phagocyte NADPH-oxidase. Studies with flavin analogues as active site probes in triton X-100-solubilized preparations

NADPH-oxidase of stimulated human neutrophil membranes was solubilized in Triton X-100 and activity reconstituted with FAD, 8-F-FAD, 8-phenyl-S-FAD, and 8-S-FAD. The enzyme had similar affinities for all the flavins with Km values in the 60-80 nM range. Vmax was found to increase 4-fold with increasing redox midpoint potential of the flavin. 8-F-FAD reconstituted with the enzyme was reactive toward thiophenol, suggesting exposure of the 8-position to solvent, a finding supported by unsuccessful attempts to label the enzyme with the photoaffinity probe 8-N3-[32P]FAD. Solubilized oxidase stabilized the red thiolate form of 8-S-FAD, a characteristic of flavoproteins of the dehydrogenase/electron transferase classes which stabilize the blue neutral form of the flavin semiquinone radical.     

Properties of D-amino-acid oxidase from Rhodotorula gracilis

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.     

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.     

Common ancestry of Escherichia coli pyruvate oxidase and the acetohydroxy acid synthases of the branched-chain amino acid biosynthetic pathway

A number of enzymes require flavin for their catalytic activity, although the reaction catalyzed involves no redox reaction. The best studied of these enigmatic nonredox flavoproteins are the acetohydroxy acid synthases (AHAS), which catalyze early steps in the synthesis of branched-chain amino acids in bacteria, yeasts, and plants. Previously, work from our laboratory showed strong amino acid sequence homology between these enzymes and Escherichia coli pyruvate oxidase, a classical flavoprotein dehydrogenase that catalyzes the decarboxylation of pyruvate to acetate. We have now shown this homology (i) to also be present in the DNA sequences and (ii) to represent functional homology in that pyruvate oxidase has AHAS activity and a protein consisting of the amino-terminal half of pyruvate oxidase and the carboxy-terminal half of E. coli AHAS I allows native E. coli AHAS I to function without added flavin. The hybrid protein contains tightly bound flavin, which is essential for the flavin substitution activity. These data, together with the sequence homologies and identical cofactors and substrates, led us to propose that the AHAS enzymes are descended from pyruvate oxidase (or a similar protein) and, thus, that the flavin requirement of the AHAS enzymes is a vestigial remnant, which may have been conserved to play a structural rather than a chemical function.     

Light-mediated reduction of flavoproteins with flavins as catalysts

It has been found that small amounts of free flavins greatly accelerate the photochemical reduction of flavoproteins both to the radical and fully reduced oxidation states. This catalytic effect has been shown to be due to the rapid photochemical reduction of the free flavin to its fully reduced state, followed by its reaction with the flavoprotein to yield flavoprotein radical and by its reaction with flavoprotein radical to yield fully reduced flavoprotein. Evidence is presented that the same route may occur with flavoproteins in the absence of added flavins. In this case the photoreduction is mediated by the small equilibrium concentration of free flavin coenzyme present in a flavorprotein solution. Hence, it is suggested that flavoprotein reduction with EDTA as photosubstrate does not involve an excited state of the holoprotein, nor contact of EDTA with the enzyme, but exchange of electrons between enzyme flavin and free reduced flavin.     

4-Thioflavins as active site probes of flavoproteins. General properties

4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents, with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at approximately 485 nm while that of the anion is approximately 425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with O2, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4-oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes.     

A near-infrared investigation of cytochrome c oxidase in higher plant mitochondria

Optical features of cytochrome c oxidase in potato mitochondria have been characterized in the near-ir region. In order to discriminate the respective properties of the various redox centers, the redox state was monitored from free and inhibited, bound species. Appropriate comparisons singled out difference spectra which can be attributed specifically to CuA and CuB. The CuA difference spectrum (red-ox) exhibits a negative band centered at 812 nm and, analogous to its mammalian counterpart, the so-called 830-nm band (delta epsilon red/ox = -2.0 mM-1 cm-1). The unusual difference spectrum (red-ox) assigned to CuB is characterized by a broad positive band also centered at 812 nm with an extinction coefficient of delta epsilon red/ox = 4.3 mM-1 cm-1.     

8-thiocyanatoflavins as active-site probes for flavoproteins.

8-Thiocyanatoflavins at the riboflavin, FMN, and FAD level were prepared via the diazonium salt of the corresponding 8-aminoflavin and some of the physical and chemical properties studied. 8-Thiocyanatoriboflavin has a UV-visible spectrum similar to that of the native flavin with absorbance maxima at 446 nm (epsilon = 14,900 M-1 cm-1) and 360 nm. Reaction with thiols such as dithiothreitol and mercaptoethanol gives rise to an 8-mercapto- and an 8-SR-flavin, whereas reaction with sulfide yields only the 8-mercaptoflavin. The 8-SCN-flavin binds to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-Old Yellow Enzyme, and apo-lactate oxidase as the FMN derivative, and to apo-D-amino acid oxidase, apo-p-hydroxybenzoate hydroxylase, apo-glucose oxidase, apo-anthranilate hydroxylase, and apo-general acyl-CoA dehydrogenase as the FAD derivative. In two cases, namely, with anthranilate hydroxylase and D-amino acid oxidase, the 8-SCN-FAD was spontaneously and completely converted to the 8-mercapto-FAD derivative, suggesting the presence of a nucleophile (most likely the thiol of a cysteine residue) in the vicinity of the 8-position. It was also found that flavodoxin stabilizes the neutral radical and Old Yellow Enzyme the anionic radical of 8-SCN-FMN. Further studies with Old Yellow Enzyme, established that fully (two electron) reduced 8-SCN-FMN undergoes photoelimination of cyanide.     

Purification and biochemical characterization of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough).

A novel iron-sulfur protein has been isolated from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough). It is a stable monomeric protein, which has a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. Analysis of the metal and acid-labile sulfur content of the protein revealed the presence of 6.3 +/- 0.4 Fe/polypeptide and 6.2 +/- 0.7 S2-/polypeptide. Non-iron transition metals, heme, flavin and selenium were absent. Combining these data with the observation of a very anisotropic S = 1/2 [6Fe-6S]3+ prismane-like EPR signal in the dithionite-reduced protein, we believe that we have encountered the first example of a prismane-cluster-containing protein. The prismane protein has a slightly acidic amino acid composition and isoelectric point (pI = 4.9). The ultraviolet/visible spectrum is relatively featureless (epsilon 280 = 81 mM-1.cm-1, epsilon 400 = 25 mM-1.cm-1, epsilon 400,red = 14 mM-1.cm-1). The shape of the protein is approximately globular (S20.w = 4.18 S). The N-terminal amino acid sequence is MFS/CFQS/C QETAKNTG. Polyclonal antibodies against the protein were raised. Cytoplasmic localization was inferred from subcellular fractionation studies. Cross-reactivity of antibodies against this protein indicated the occurrence of a similar protein in D. vulgaris (Monticello) and Desulfovibrio desulfuricans (ATCC 27774). We have not yet identified a physiological function for the prismane protein despite trials for some relevant enzyme activities.     

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.     

Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by alpha-lipoamide dehydrogenase flavin (Em7.4 = -283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme (Em7.4 = -52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, alpha-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.     

Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.     

The acceptor specificity of flavins and flavoproteins. III. Flavoproteins

1. The specificity of flavoproteins towards acceptors has been rather neglected, but an attempt is here made to construct a comparative table of acceptor specificities of those flavoprotein enzymes for which data exist.

2. The acceptor specificity of reduced flavin groups, when combined with apoenzyme proteins, is quite different from that of the same flavin groups in the free state (see Part II). Free flavins react very rapidly with a wide range of acceptors, but the same groups combined as flavoproteins have a severely restricted range of action.

3. There are remarkable differences between different flavoproteins. Nearly every flavoprotein fails altogether to react with at least one, and often several, of the acceptors, giving a specificity pattern which is different in each case. There seems to be no general acceptor for flavoproteins.

4. The effect of combination of a flavin with a particular apoenzyme is to inhibit specifically the reaction of the flavin with particular acceptors with which it would react very rapidly in the absence of the apoenzyme.

5. Each apoenzyme produces its own distinctive pattern of inhibitions. The degree of inhibition is often very high; the table shows over 50 cases of specific inhibitions that are essentially complete. Some of these are very difficult to explain.

6. There is no obvious parallelism between any acceptor and any other in its pattern of reactivity with a series of different flavoproteins.

7. In a few cases combination with apoenzyme specifically accelerates the reaction of the flavin with particular acceptors, so that the flavoprotein is oxidized faster than the free flavin.

8. Possible correlations are discussed between the effects of apoenzymes on the reactivity of flavins with acceptors and a number of special known features of different apoenzymes, but no adequate explanation of the differences in specificity has emerged.

9. In view of the interesting nature of the effects, a plea is made for a more intensive study of the acceptor side of flavoprotein specificity.     

Electron transfer flavoprotein from pig liver mitochondria. A simple purification and re-evaluation of some of the molecular properties.

Electron transfer flavoprotein (ETF) from pig liver mitochondria has been purified to homogeneity by a three-step procedure with approx. 10-fold higher yields than previously reported. The purified ETF exhibits an absorption coefficient for the bound FAD of 13.5 mM-1.cm-1 at 436 nm and an isoelectric point of 6.75. Gel filtration, sodium dodecyl sulphate gel electrophoresis and flavin analysis indicate that pig liver ETF is a dimer, composed of non-identical subunits (Mr 38 000 and 32 000) with only one FAD/dimer. Anaerobic reduction by dithionite produces anionic flavin semiquinone as a stable intermediate and establishes the flavin to be the only redox-active chromophore in ETF.     

Oxygen activation in flavoprotein oxidases: the importance of being positive

The oxidation of flavin hydroquinones by O(2) in solution is slow, with second-order rate constants of ~250 M(-1) s(-1). This is due to the obligatory, single-electron transfer that initiates the reaction being thermodynamically unfavored and poorly catalyzed. Notwithstanding considerations of O(2) accessibility to the reaction site, its desolvation and geometry and other factors that can also contribute to further rate acceleration, flavoprotein oxidases must activate O(2) for reaction with flavin hydroquinones to be able to achieve the 100-1000-fold rate enhancements typically observed. Protein positive charges have been identified in glucose oxidase, monomeric sarcosine oxidase, N-methyltryptophan oxidase and fructosamine oxidase that electrostatically stabilize the transition state for the initial single electron transfer that generates the O(2)(-?)/flavin semiquinone radical pair. In choline oxidase despite the presence of three histidines in the active site, the trimethylammonium group of the reaction product provides such an electrostatic stabilization. A nonpolar site proximal to the flavin C(4a) atom in choline oxidase has also been identified, which contributes to the geometry and desolvation of the O(2) reaction site. The relevance of O(2) activation by product charges to other flavoprotein oxidases, such as for example those catalyzing amine oxidations, is discussed in this review. A nonpolar site close to the flavin C(4a) atom and a positive charge is identified through structural analysis in several flavoprotein oxidases. Mutagenesis has disclosed nonpolar sites in O(2)-reducing enzymes that utilize copper/TPQ or iron. It is predicted that classes of O(2)-reducing enzymes utilizing other cofactors also contain a similar catalytic motif.     

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+.