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PubMed2002

The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum.

The endoplasmic reticulum (ER) supports disulfide formation through an essential protein relay involving Ero1p and protein disulfide isomerase (PDI). We find that in addition to having a tightly associated flavin adenine dinucleotide (FAD) moiety, yeast Ero1p is highly responsive to small changes in physiological levels of free FAD. This sensitivity underlies the dependence of oxidative protein folding on cellular FAD levels. FAD is synthesized in the cytosol but can readily enter the ER lumen and promote Ero1p-catalyzed oxidation. Ero1p then uses molecular oxygen as its preferred terminal electron acceptor. Thus Ero1p directly couples disulfide formation to the consumption of molecular oxygen, but its activity is modulated by free lumenal FAD levels, potentially linking disulfide formation to a cell's nutritional or metabolic status.

Blotting, Western↗
PubMed2003

Structural characterization and mapping of the covalently linked FAD cofactor in choline oxidase from Arthrobacter globiformis.

The flavoenzyme choline oxidase catalyzes the oxidation of choline and betaine aldehyde to betaine. Earlier studies have shown that the choline oxidase from Arthrobacter globiformis contains FAD covalently linked to a histidine residue. To identify the exact type of flavin binding, the FAD-carrying amino acid residue was released by acid hydrolysis. The fluorescence excitation maxima of the isolated aminoacylriboflavin, showing a hypsochromic shift of the near-ultraviolet band relative to riboflavin, and the pH-dependent flavin fluorescence confirmed the presence of an 8alpha-substituted flavin linked to histidine. Similarly, MALDI-TOF mass spectrometry showed a molecular mass corresponding to histidylriboflavin. Classical experiments used to distinguish between the N(1) and N(3) isomers all indicated that the flavin was linked to the N(1) position of the histidine residue. The position of the FAD-carrying histidine residue in the choline oxidase polypeptide was identified by tryptic cleavage of the denatured enzyme, HPLC separation of the proteolytic peptide fragments, and characterization of the purified flavin-carrying peptide by mass spectrometry and spectroscopy. The FAD moiety was assigned to the tryptic peptide, His-Ala-Arg, corresponding to residues 87-89 in the open reading frame of the previously published cDNA sequence. Further analysis of the flavopeptide by collision-induced dissociation mass spectrometry confirmed that the flavin cofactor was attached to His(87). We conclude that this variant of choline oxidase contains 8alpha-[N(1)-histidyl]FAD at position 87 in the polypeptide chain.

Alcohol Oxidoreductases↗
PubMed2005

The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

Base Sequence↗
PubMed2006

Mechanism of FAD reduction and role of active site residues His-225 and Tyr-259 in Arthrobacter globiformis dimethylglycine oxidase: analysis of mutant structure and catalytic function.

Residues His-225 and Tyr-259 are located close to the FAD in the dehydrogenase active site of the bifunctional dimethylglycine oxidase (DMGO) of Arthrobacter globiformis. We have suggested [Leys, D., Basran, J., and Scrutton, N. S. (2003) EMBO J. 22, 4038-4048] that these residues are involved in abstraction of a proton from the substrate amine group of dimethylglycine prior to C-H bond breakage and FAD reduction. To investigate this proposal, we have isolated two mutant forms of DMGO in which (i) His-225 is replaced with Gln-225 (H225Q mutant) and (ii) Tyr-259 is replaced with Phe-259 (Y259F mutant). Both mutant enzymes retain the ability to oxidize substrate, but the steady-state turnover of the Y259F mutant is attenuated more than 200-fold. Only modest changes in kinetic parameters are observed for the H225Q mutant during steady-state turnover. Stopped-flow studies indicate that the rate of FAD reduction in the Y259F enzyme is substantially impaired by a factor of approximately 1500 compared with that of the wild-type enzyme, suggesting a key role for this residue in the reductive half-reaction of the enzyme. The kinetics of FAD reduction in the H225Q enzyme are complex and involve three discrete kinetic phases that are attributed to different conformational states of this mutant, evidence for which is provided by crystallographic analysis. Neither the H225Q enzyme nor the Y259F enzyme stabilizes the FADH(2)-iminium charge-transfer complex observed previously in stopped-flow studies with the wild-type enzyme. Our studies are consistent with a key role for Tyr-259, but not His-225, in deprotonation of the substrate amine group prior to FAD reduction. We infer that residue His-225 is likely to modulate the acid-base properties of Tyr-259 by perturbing the pK(a) of Tyr-259 and thus fine-tunes the reaction chemistry to facilitate proton abstraction under physiological conditions. Our data are discussed in the context of the crystallographic data for DMGO and also in relation to contemporary mechanisms for flavoprotein-catalyzed oxidation of amine substrates.

Arthrobacter↗
PubMed1998

Proposed steady-state kinetic mechanism for Corynebacterium ammoniagenes FAD synthetase produced by Escherichia coli.

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).

Adenosine Triphosphate↗
PubMed1999

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester.

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K(d) 6.7 x 10(-7) M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavin-binding site of the enzyme, apo-L-aspartate oxidase has been reacted with N6-(6-carboxyhexyl)-FAD succinimidoester. The structural characterization of the resulting N6-(6-carbamoylxyhexyl)FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/monomer. Residue Lys38 was identified as the target of the covalent modification. N6-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.

Amino Acid Oxidoreductases↗
PubMed1991

Hexose metabolism in pancreatic islets. Glucose-induced and Ca(2+)-dependent activation of FAD-glycerophosphate dehydrogenase.

A rise in extracellular D-glucose concentration causes in pancreatic islets a preferential stimulation of aerobic, relative to total, glycolysis. The possible participation in such a phenomenon of a glucose-induced and Ca(2+)-dependent activation of FAD-glycerophosphate dehydrogenase was investigated. In islet homogenates, the activity of the mitochondrial and Ca(2+)-responsive FAD-glycerophosphate dehydrogenase was about two orders of magnitude lower than that of the cytosolic and Ca(2+)-insensitive NAD-glycerophosphate dehydrogenase. In islet mitochondria, Ca2+ increased the affinity of the FAD-glycerophosphate dehydrogenase for L-glycerol 3-phosphate, but did not affect the maximal reaction velocity. In the presence of 0.1 mM-L-glycerol 3-phosphate, the Ka for Ca2+ was close to 0.1 microM. When intact islets were preincubated in the presence of both D-glucose and Ca2+, the activity of FAD-glycerophosphate dehydrogenase measured in intact mitochondria incubated in the presence of 1.2 microM-Ca2+ was higher than that recorded under the same conditions in islets preincubated in the absence of D-glucose and/or Ca2+. These findings support the view that, in islets exposed to a high concentration of D-glucose, a Ca(2+)-induced activation of mitochondrial FAD-glycerophosphate dehydrogenase favours the transfer of reducing equivalents by the glycerol phosphate shuttle, and hence accounts, in part at least, for the preferential stimulation of aerobic glycolysis.

Animals↗
PubMed1995

Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments.

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

Adenosine Monophosphate↗
PubMed1996

A requirement for matrix processing peptidase but not for mitochondrial chaperonin in the covalent attachment of FAD to the yeast succinate dehydrogenase flavoprotein.

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial heterotetramer containing a flavoprotein subunit with an 8alpha-N(3)-histidyl-linked FAD cofactor. The covalent linkage of the FAD is necessary for activity. We have developed an in vitro assay that measures the flavinylation of the flavoprotein precursor in mitochondrial matrix fractions. Flavoprotein modification does not depend on translocation across a membrane, but it does require proteolytic processing by the mitochondrial processing peptidase prior to flavin attachment. Since ATP depletion, N-ethylmaleimide, or proteinase treatments of matrix fractions inhibit flavoprotein modification, at least one additional matrix protein component appears to be required. Having previously suggested that the flavoprotein begins folding before FAD attachment occurs, we tested whether the mitochondrial chaperonin, heat shock protein 60, might be necessary. Co-immunoprecipitation of the flavoprotein and the chaperonin demonstrate that the proteins do indeed interact. However, immunodepletion of the chaperonin from matrix fractions does not inhibit FAD attachment. Nonprotein components are also required for flavoprotein modification. In addition to ATP, effector molecules such as succinate, fumarate, or malate also stimulate modification. Together, these results suggest that FAD addition is an early event in succinate dehydrogenase assembly.

Adenosine Triphosphate↗
PubMed1999

Cellobiose dehydrogenase from the fungi Phanerochaete chrysosporium and Humicola insolens. A flavohemoprotein from Humicola insolens contains 6-hydroxy-FAD as the dominant active cofactor.

Cellobiose dehydrogenases (CDH) were purified from cellulose-grown cultures of the fungi Phanerochaete chrysosporium and Humicola insolens. The pH optimum of the cellobiose-cytochrome c oxidoreductase activity of P. chrysosporium CDH was acidic, whereas that of H. insolens CDH was neutral. The absorption spectra of the two CDHs showed them to be typical hemoproteins, but there was a small difference in the visible region. Limited proteolysis between the heme and flavin domains was performed to investigate the cofactors. There was no difference in absorption spectrum between the heme domains of P. chrysosporium and H. insolens CDHs. The midpoint potentials of heme at pH 7.0 were almost identical, and no difference in pH dependence was observed over the range of pH 3-9. The pH dependence of cellobiose oxidation by the flavin domains was similar to that of the native CDHs, indicating that the difference in the pH dependence of the catalytic activity between the two CDHs is because of the flavin domains. The absorption spectrum of the flavin domain from H. insolens CDH has absorbance maxima at 343 and 426 and a broad absorption peak at 660 nm, whereas that of P. chrysosporium CDH showed a normal flavoprotein spectrum. Flavin cofactors were extracted from the flavin domains and analyzed by high-performance liquid chromatography. The flavin cofactor from H. insolens was found to be a mixture of 60% 6-hydroxy-FAD and 40% FAD, whereas that from P. chrysosporium CDH was normal FAD. After reconstitution of the deflavo-proteins it was found that flavin domains containing 6-hydroxy-FAD were clearly active but their cellobiose oxidation rates were lower than those of flavin domains containing normal FAD. Reconstitution of flavin cofactor had no effect on the optimum pH. From these results, it is concluded that the pH dependence is not because of the flavin cofactor but is because of the protein molecule.

Carbohydrate Dehydrogenases↗
PubMed2000

Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A.

The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins.

Binding Sites↗
PubMed1985

Characterization of fumarate reductase from baker's yeast: essential sulfhydryl group for binding of FAD.

Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.

Apoenzymes↗
PubMed1975

Stimulation of erythrocyte glutathione reductase activity by flavin adenine dinucleotide (FAD) in Malaysian adults and newborns and their parents.

A study of glutathione reductase (GR) activity and its stimulation by flavin adenine dinucleotide (FAD) in erythrocytes of Malaysian newborns and adults of different racial groups showed that GR stimulation by FAD was greater than 20% in 50% of 866 newborns (57% of Malays, 55% of Indians and 41% of Chinese) and 54% of 274 adults (46% of Malays, 65% of Indians and 45% of Chinese). There was a significant negative correlation between GR activity and percentage FAD stimulation in both newborns and adults in all racial groups. Low GR activity and a high percentage FAD stimulation were more prevalent among parents of newborns with low GR activity than among parents of newborns with higher GR activity. Administration of riboflavin to mothers with low GR activity resulted in increased GR activity and a decreased percentage of FAD stimulation. None of the individuals examined had clear clinical manifestations of riboflavin deficiency. It is concluded that subclinical riboflavin deficiency leading to low GR activity is prevalent in Malaysia among adults and newborns, especially among Malays and Indians.

Adult↗
PubMed1997

The Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus--redox states of the FAD prosthetic group and mechanism of Ag+ inhibition.

The FAD prosthetic group of the Na+-motive NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio alginolyticus was investigated by ultraviolet-visible and fluorescence spectroscopy. The reduction of Na+-NQR by excess NADH in the presence of 6-13 microM O2 resulted in the formation of the blue flavosemiquinone radical. If the concentration of dioxygen was further reduced to 0.1 microM O2, neither the reduction of Na+-NQR by NADH nor its reoxidation with ubiquinone-1 (Q-1) yielded a stable flavosemiquinone in equilibrium with reductant or oxidant, respectively, but the fully reduced (Fl(red)H2) or oxidized flavin (Fl(ox)) prevailed. During reoxidation of Fl(red)H2 with Q-1, the intermediate formation of an absorbance band around 800 nm was observed, which was tentatively assigned as the Fl(red)H(-)-NAD+ charge-transfer complex. Complete reoxidation of Fl(red)H2 in Na+-NQR was achieved by a fivefold excess of Q-1 over NADH. These results indicated that only a small fraction of FAD was in the flavosemiquinone redox state during turnover to mediate the electron transfer between the hydride donor, NADH, and the one-electron acceptor [2Fe-2S]. The titration of Na+-NQR with Ag+, a specific inhibitor, was followed by the fluorescence emission spectra of FAD (Fl(ox)). The addition of Ag+ resulted in a marked increase of the flavin fluorescence (16% at 200 nM Ag+), with half-maximal saturation at approximately 50 nM Ag+, indicating dissociation of FAD from the enzyme. The increase in fluorescence intensity correlated with the loss of enzyme activity. Gel filtration of the Ag+-treated Na+-NQR confirmed that FAD had been displaced from the holo-enzyme.

Bacterial Proteins↗
PubMed2005

Effects of RNAi-mediated silencing of PEN-2, APH-1a, and nicastrin on wild-type vs FAD mutant forms of presenilin 1.

The gamma-secretase complex consists of PS1/PS2, nicastrin, APH-1a, and PEN-2. PS1 undergoes endoproteolytic processing to yield two fragments: PS1-NTF and PS1-CTF. Changes in PEN-2 levels have been shown previously to affect the endoproteolytic processing of wild-type (wt)-PS1. However, the effects of PEN-2 on the proteolytic processing of familial Alzheimer's disease (FAD) mutant forms of PS1 have not yet been reported. To determine whether PEN-2 affects the proteolytic processing of mutant PS1 in the same manner as that of wt-PS1, we established RNA interference (RNAi) for PEN-2 in H4 human neuroglioma cells stably transfected to express wt or FAD mutant forms of PS1 including L286V, A246E, and that lacking exon 9 (Delta9). As expected, in H4 cells expressing wt-PS1, RNAi for PEN-2 increased levels of PS1-FL and attenuated PS1 endoproteolysis. Likewise, in cells expressing PS1 with the FAD missense mutations, L286V and A246E, RNAi for PEN-2 increased PS1-FL and reduced PS1 endoproteolysis. However, in H4 cells stably transfected to express the FAD-linked Delta9 mutation (PS1 lacking exon 9), RNAi for PEN-2 did not increase but, instead, decreased PS1-FL. In contrast, RNAi for nicastrin and APH-1a decreased PS1-FL in H4 cells expressing either wt-PS1 or Delta9-PS1. In summary, the metabolism of wt-PS1 and FAD-linked Delta9-PS1 is specifically and differentially affected by loss of function of PEN-2.

Alzheimer Disease↗
PubMed1988

Stereospecificity in the oxidation of phorate and phorate sulphoxide by purified FAD-containing mono-oxygenase and cytochrome P-450 isozymes.

1. Both the cytochrome P-450-dependent mono-oxygenase system and the FAD-containing mono-oxygenase catalyse the sulphoxidation of thioether-containing organophosphate insecticides. Using purified FAD-containing mono-oxygenase and purified cytochrome P-450 isozymes isolated from mouse liver microsomes, the stereospecificity of the oxidation of phorate to (+)-and (-)-phorate sulphoxide and the further oxidations of the (+)-and (-)-phorate sulphoxides to the sulphone, the oxon sulphoxide and the oxon sulphone were examined. 2. The FAD-containing mono-oxygenase catalysed the formation of (-)-phorate sulphoxide, while two cytochrome P-450 isozymes (cytochrome P-450-B2, a constitutive form, and cytochrome P-450-PB, the principal form induced by phenobarbital) produced (+)-phorate sulphoxide. The other three constitutive cytochrome P-450 isozymes examined yielded racemic mixtures. 3. The FAD-containing mono-oxygenase had the lowest Km for the sulphoxidation reaction, 32 microM, while the Km values for the cytochrome P-450 isozymes ranged from 67 microM to 250 microM. No additional oxidation of phorate sulphoxide by the FAD-containing monooxygenase was detected using either (+)-phorate sulphoxide or (-)-phorate sulphoxide as substrates. 4. In contrast, all five cytochrome P-450 isozymes tested formed additional oxidation products; the (+)-phorate sulphoxide was the preferred substrate for all cytochrome P-450 forms. 5. The final oxidation product, phorate oxon sulphone, was derived by desulphuration of phorate sulphone, with the formation of the oxon sulphoxide being a terminal pathway.

Animals↗
PubMed1977

Effect of halide anions on the binding of FAD to D-amino acid oxidase and the tryptophanyl fluorescence of the apoenzyme.

The quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25 degrees C. All of the tryptophanyl fluorescence of the apoenzyme in 6 M guanidine hydrochloride was quenched. The tryptophanyl fluorescence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism. By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution containing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.

Animals↗
PubMed1985

An essential cysteine residue located in the vicinity of the FAD-binding site in short-chain, medium-chain, and long-chain acyl-CoA dehydrogenases from rat liver mitochondria.

Medium-chain and long-chain acyl-CoA dehydrogenases from rat liver have been purified in two forms, holoenzymes containing FAD and apoenzymes which do not contain this cofactor. In contrast, short-chain acyl-CoA dehydrogenase can only be isolated as the holoenzyme. Marked differences in the reactivity to organic sulfhydryl reagents were observed between the apo and holo forms of these enzymes. While the two apoenzymes were severely inactivated by N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB), and iodoacetate (IAA), the two corresponding holoenzymes were not susceptible to these reagents. The inactivation of the two apoenzymes by NEM followed pseudo-first order kinetics. Incubation of the apoenzymes with FAD completely prevented the inactivation by the organic sulfhydryl reagents. Methylmercury halides (iodide or chloride) inactivated both the apo and holo forms of medium-chain and long-chain acyl-CoA dehydrogenases. On the other hand, holo-short-chain acyl-CoA dehydrogenase behaved somewhat differently from the other two holoenzymes in that it was inactivated by pCMB (but not NEM or IAA) following a pseudo-first order process. The titration of the two apoenzymes with [14C]NEM and that of the holo-short-chain acyl-CoA dehydrogenase with [14C]pCMB indicated that all three acyl-CoA dehydrogenases contain a single essential cysteine residue/subunit. In the inactivation of holo-medium-chain and holo-long-chain acyl-CoA dehydrogenases with methylmercury halide, the same essential cysteine residue was modified without perturbing or releasing the enzyme-bound FAD. The inactivations of the three holoenzymes by appropriate organic sulfhydryl reagents were prevented by prior incubation with substrate. These experimental results indicate that the essential cysteine residue is located in the vicinity of the FAD- and substrate-binding sites within the active center of the enzymes. It appears, however, that this cysteine residue does not participate directly in FAD binding.

Acyl-CoA Dehydrogenase, Long-Chain↗